s2fieldsofstudy: Biology Medicine

text:
Impact of intensification with raltegravir on HIV-1-infected individuals receiving monotherapy with boosted PIs

Abstract Background Monotherapy with ritonavir-boosted PIs (PI/r) has been used to simplify treatment of HIV-1-infected patients. In previous studies raltegravir intensification evidenced ongoing viral replication and reduced T cell activation, preferentially in subjects receiving PI-based triple ART. However, data about low-level viral replication and its consequences in patients receiving PI/r monotherapy are scarce. Methods We evaluated the impact of 24 weeks of intensification with raltegravir on markers of viral persistence, cellular immune activation and inflammation biomarkers in 33 patients receiving maintenance PI/r monotherapy with darunavir or lopinavir boosted with ritonavir. ClinicalTrials.gov identifier: NCT01480713. Results The addition of raltegravir to PI/r monotherapy resulted in a transient increase in 2-LTR (long-terminal repeat) circles in a significant proportion of participants, along with decreases in CD8+ T cell activation levels and a temporary increase in the expression of the exhaustion marker CTLA-4 in peripheral T lymphocytes. Intensification with raltegravir also reduced the number of samples with intermediate levels of residual viraemia (10–60 HIV-1 RNA copies/mL) compared with samples taken during PI/r monotherapy. However, there were no changes in cell-associated HIV-1 DNA in peripheral CD4+ T cells or soluble inflammatory biomarkers (CD14, IP-10, IL-6, C-reactive protein and D-dimer). Conclusions Intensification of PI/r monotherapy with raltegravir revealed persistent low-level viral replication and reduced residual viraemia in some patients during long-term PI/r monotherapy. The concomitant change in T cell phenotype suggests an association between active viral production and T cell activation. These results contribute to understanding the lower efficacy rates of PI/r monotherapies compared with triple therapies in clinical trials.

Introduction
Combined ART has significantly improved the life expectancy of HIV-1-infected individuals. Current international guidelines recommend first-line therapy consisting of three drugs from at least two different classes, which generally includes two NRTIs plus a third agent. This combination decreases plasma viral load to undetectable levels by standard assays (,50 HIV-1 RNA copies/mL) and suppresses viral replication for as long as treatment is maintained. Thus, life-long ART is needed to prevent the development of drug resistance and viral recrudescence. However, extended use of ART is expensive and may lead to long-term complications such as hyperlipidaemia, decreased bone mineral density, renal toxicity and cardiovascular diseases. [1][2][3][4] Hence, interest in ART simplification strategies [e.g. monotherapy with ritonavir-boosted PIs (PI/r)] as maintenance therapy for individuals who have already achieved viral suppression with triple ART has grown in recent years. 5 Current ART cannot eradicate HIV-1 infection, because viral reservoirs remain in the form of silent HIV-1 DNA integrated in the host genome of long-lived resting memory CD4! T cells. Indeed, the use of novel ultrasensitive procedures has shown that residual plasma viraemia (,50 copies HIV-1 RNA/mL) is present in a significant proportion of individuals on ART in whom viral replication has been well-suppressed for years. 6 However, the origin of this residual viral production during ART remains unknown.
Intensification of ART with raltegravir has been assayed in several clinical trials in an attempt to completely suppress residual viraemia. Although this primary endpoint was not achieved, some studies detected a transient increase in episomes containing two copies of the viral long-terminal repeat (2-LTR circles). 7,8 Given that integrase inhibitors block viral linear DNA from integration into genomic host cell DNA, leading to the accumulation of circular viral DNA molecules (including 2-LTR circles) in recently infected cells, the increase in viral episomes observed after intensification with raltegravir in some individuals suggests the persistence of lowlevel viral replication during ART. Interestingly, in both studies, the increase in the level of 2-LTR circles after intensification with raltegravir was more frequent in patients receiving a PI-containing ART regimen. Although active replication may occur in compartments that are less accessible to PIs, 9 regimens consisting of three reverse transcriptase inhibitors might also reduce the probability of formation of the linear DNA precursor to episomes. 7 In addition, studies evaluating intensification with raltegravir have also shown a significant decrease in levels of CD8! T cell activation. 7,10,11 Taken together, these observations suggest that PI/r monotherapy might be even less effective at completely suppressing low-level viral replication than PI-containing triple ART.
Here, we report the results of a pilot trial in which individuals on maintenance monotherapy with PI/r had their treatment temporarily intensified with raltegravir for 24 weeks. We evaluated the impact of intensification on low-level viral replication, viral reservoirs, cellular immune activation and soluble markers of inflammation.

Ethics
We performed a proof-of-concept, single-arm, pilot clinical trial (ClinicalTrials.gov identifier: NCT01480713; EudraCT number 2011-004464-30) at the Hospital Universitari Germans Trias i Pujol (Badalona, Spain). The study was conducted in compliance with the principles of the Declaration of Helsinki and in accordance with the Spanish legislation. The Institutional Ethics Review Committee approved the protocol (approval number AC-11-083) and all participants provided their written informed consent.

Study design and participants
Sample size was established on the basis of previous data on intensification with raltegravir in participants receiving PI-containing regimens. 7 We recruited 33 HIV-1-infected adults with undetectable plasma viraemia (,50 HIV-1 RNA copies/mL) who had been on fully suppressive PI/r monotherapy (400/100 mg of lopinavir/ritonavir twice daily or 800/100 mg of darunavir/ritonavir once daily) for at least the previous 12 months and had switched from triple ART to PI/r monotherapy while on viral suppression. Other inclusion criteria were an absolute CD4! T cell count of 500 cells/mm 3 and absence of virological failure to previous PI-containing regimens. Adherence to treatment was reported by the participants using the adapted SERAD (Self-Reported Adherence) questionnaire. 12 Adherence was further verified by monitoring antiretroviral plasma drug levels during the intensification period and at the end of the follow-up.
Three blood samples were collected during the 2 months preceding initiation of raltegravir (Figure 1a). During the intensification period, samples were collected at weeks 1, 2, 4, 8, 12 and 24. Two additional samples were collected 12 and 24 weeks after withdrawal of raltegravir (i.e. weeks 36 and 48 of the study).

Quantification of cell-associated HIV DNA
CD4! T cells were purified from 20%10 6 cryopreserved PBMCs by negative immunomagnetic separation (CD4! T Cell Isolation Kit, Milteny). Total HIV-1 DNA was quantified by Droplet Digital PCR (ddPCR; Bio-Rad) from lysed extracts, as previously described, 13 with a detection limit of 1 copy/million CD4! T cells. The same methodology was used to measure 2-LTR circles (forward primer, 5 0 -CTAACTAGGGAACCCACTGCT-3 0 ; reverse primer, 5 0 -GTAGT TCTGCCAATCAGGGAA-3 0 ; probe, 5 0 -AGCCTCAATAAAGCTTGCCTTGAGTGC-3 0 ). The RPP30 cellular gene was quantified to normalize sample input. All samples from the same individual were processed in parallel to minimize the effect of experimental variability on the analysis of viral dynamics. FAM/HEX-ZEN/Iowa Black FQ dual-labelled double-quenched probes were purchased from Integrated DNA Technologies.

Residual viraemia
Throughout the study period, participants' viral load was monitored every 3 months using the Abbott RealTime HIV-1 assay (Abbott Molecular Inc.). In order to quantify plasma viraemia under the detection limit of standard methodologies (50 HIV-1 RNA copies/mL), cryopreserved plasma samples were analysed using an ultrasensitive viral load test. 13 Briefly, up to 7.5 mL of plasma was ultracentrifuged prior to extraction and quantification of viral RNA using the Abbott RealTime HIV-1 assay and the Abbott m2000rt instrument. An in-house calibration curve set (range 10 1 -10 3 copies/mL), which had previously been validated using a standard HIV-1 RNA control from the WHO, was used as a reference.

Soluble markers
Plasma levels of several markers of inflammation and coagulation were measured in frozen plasma samples. D-dimer and C-reactive protein (CRP) were measured using an aggregation assay (Biokit). Soluble CD14 (sCD14), IFN-c-induced protein 10 (IP-10) and IL-6 were measured using ELISA following the manufacturer's instructions (Diaclone).

Statistical analysis
Longitudinal analysis of residual viraemia, with data below the limit of detection, was first performed by comparing timepoints using the paired Prentice-Wilcoxon test. To evaluate the difference in the frequency of intermediate viraemia levels (.10 HIV-1 RNA copies/mL) during raltegravir intensification and after drug withdrawal, compared with the preintensification period, a mixed-effects logistic regression model was fitted for the binary outcome indicative of intermediate residual viraemia, assuming constant probabilities of exceeding 10 copies of residual viraemia for each patient in each period. Longitudinal comparisons between subjects with detectable 2-LTR circles (2-LTR!) and individuals with undetectable 2-LTR circles (2-LTR#) were made using piecewise linear mixed-effects models. Inference on the distribution of the 2-LTR peaks was assessed by calculating an empirical P value through a Monte Carlo simulation. Comparisons between timepoints for the levels of cell-associated HIV-1 DNA, T cell immunophenotype and inflammation markers were made using the Wilcoxon signed rank test. The statistical analysis was performed using R and GraphPad Prism 5.

Participant characteristics and disposition
Thirty-three participants were enrolled in the raltegravir intensification protocol (400 mg twice daily). Of these, 18 individuals (54.5%) were receiving darunavir/ritonavir and 15 (45.5%) lopinavir/ritonavir. The baseline characteristics are summarized in Table 1. Most participants were men (81.8%) and seven were coinfected with HCV. Median time on suppressive ART was 7.4 years and median time on PI/r monotherapy was 3.3 years. To evaluate the reversibility of the potential changes induced by intensification with raltegravir on viral and immune dynamics, 30 individuals were further followed beyond discontinuation of raltegravir at week 24 ( Figure 1a). Therapeutic drug monitoring indicated optimal plasma drug levels for all participants during the intensification period. Likewise, 97% of the subjects self-reported treatment adherence greater than 96%. During the study, three participants reported mild adverse effects (headache, gastrointestinal disturbances, fatigue) and one moderate event (aggressive anxiety) potentially related to ART, although none of these led to discontinuation of treatment.
Virological failure was not observed during the study. Isolated viraemia blips (preceded and followed by negative results) were detected by routine determination of viral load (Abbott RealTime HIV-1 assay) in four individuals; the blip was during the intensification period in only one case and was not confirmed by ultrasensitive determination of plasma viraemia in the same sample.

Residual viraemia
Residual viraemia was measured using 4.5-7 mL of stored plasma samples collected before intensification (Figure 1a), at weeks 4, 12 and 24 after initiation of raltegravir and also at weeks 12 and 24 after withdrawal of raltegravir [median (IQR) limit of quantification, 0.7 (0.6-0.8) HIV-1 RNA/mL]. Median (IQR) residual viraemia at baseline was 2.2 (0.0-6.6) HIV-1 RNA copies/mL and longitudinal analysis showed no significant changes in median values during intensification or after withdrawal (Figure 1b, left y-axis, and Table 2). Indeed, the proportion of participants with detectable residual viraemia (.1 HIV-1 RNA copy/mL) was similar at baseline and at the end of the intensification period (56% versus 53%). However, we observed a higher proportion of samples with intermediate residual viraemia levels (10-60 HIV-1 RNA copies/mL) in the pre-intensification period (21%) than during administration of raltegravir (7%) and a rising tendency was observed after discontinuation of raltegravir (15%) (Figure 1b, right y-axis). A mixed-effects logistic regression model revealed a significant decrease in the probability to present intermediate residual viraemia levels during the intensification period (OR " 0.16 and P " 0.01; Figure 1c). However, this effect was lost after raltegravir withdrawal (OR " 0.73 and P " 0.65). This result reflects the impact of raltegravir on residual viraemia in individuals on maintenance PI/r monotherapy.

2-LTR dynamics
To provide further insights into the efficacy of PI/r monotherapy for suppressing low-level viral replication, we explored the effect of intensifying ART with raltegravir on markers of de novo infection. For this purpose, we analysed the dynamics of 2-LTR circles in CD4! T cells, as episomal HIV-1 DNA is a surrogate marker of recent infection events. 2-LTR circles were systematically detected in 18 participants (54.5%), with a median (IQR) level of 7.75 (4.25-14.9) 2-LTR copies/million CD4! T cells at baseline. The longitudinal analysis showed a transient increase in the first 2 months following initiation of raltegravir (Figure 2a). However, no significant changes were detected at the end of the intensification period (P " 0.30 from baseline to week 24; Table 2). Individual dynamics showed that, in most participants, the highest 2-LTR level was reached between week 1 and week 8 (Figure 2b). The difference in concentrations of peaks was statistically significant (P " 0.0005). The results confirm the persistence of low-level viral replication during PI/r monotherapy, which was susceptible to further inhibition by raltegravir.    Puertas et al.

Analysis of T cell subsets, activation and exhaustion markers
Intensification with raltegravir reduces CD8! T cell activation and modestly modulates the maturation profile of CD4! T cells. 11 Both parameters were also analysed longitudinally. Although the percentage of CD4! T cells increased between baseline and week 24 (P " 0.0066) in parallel with a contraction of the CD8! T cell compartment (P " 0.0015), no significant increases in the CD4!/CD8! ratio were observed (data not shown). Furthermore, no major longitudinal changes were observed in the frequency of various CD8! T cell subpopulations (naive cells or central, transitional and effector memory subsets), while changes in CD4! T cells were associated with an increase in the transitional memory subpopulation (CD4! CD45RA# CCR7# CD27!, P " 0.045, data not shown) from baseline to week 24. No significant changes in immune activation (percentage of CD38! cells) were observed within the CD4! T cell compartment (Figure 3a). In contrast, a general decrease in the expression of this activation marker was observed in the CD8! T cell compartment during the intensification period (P " 0.0075) (Figure 3b). Interestingly, when we compared activation levels according to whether 2-LTR circles had been detected or not (2-LTR! and 2-LTR#), a slightly higher frequency of CD38! cells was observed within CD4! T cells in 2-LTR! individuals (Figure 3c). This difference reached statistical significance in the CD8! T cell population (P " 0.006) (Figure 3d). No changes were observed in the frequency of the HLA-DR! or in double CD38! HLA-DR! in CD4! or CD8! T cells (data not shown).
The analysis of the exhaustion marker CTLA-4 showed no significant changes during the study period, although a transient increase affecting all memory subsets was observed during intensification between weeks 1 and 12, both in the CD4! and in the CD8! T cell populations (Figure 4a and b). In contrast with the immune activation levels, exhaustion was similar in CD8! T cells when individuals were segregated according to detection of 2-LTRs. However, in 2-LTR! individuals, there tended to be more  Asterisks denote significant differences between timepoints detected using the Wilcoxon signed rank test (*P , 0.05, **P , 0.01, ***P , 0.001). T cell activation was compared between 2-LTR! (black continuous line) and 2-LTR# (grey broken line) subgroups within CD4! (c) and CD8! (d) T cell subsets. The median and IQR are shown. Asterisks denote significant differences between 2-LTR subgroups detected using a linear mixed-effects model (*P , 0.05, **P , 0.01, ***P , 0.001). The grey zone represents the intensification period (week 0 to 24). PI monotherapy intensified with raltegravir JAC CTLA-4! cells in the CD4! T cell compartment (P " 0.09). Intriguingly, the transient elevation in CTLA-4! cells seemed to be independent of the detection of 2-LTRs (Figure 4c and d).
Finally, the longitudinal analysis of inflammatory and coagulation markers (sCD14, IP-10, IL-6, CRP and D-dimer) showed some fluctuations during the study period (for IL-6, CRP and D-dimer); however, no significant decreases associated with intensification with raltegravir were noticed (Table S1, available as Supplementary data at JAC Online).

Discussion
Randomized clinical trials have shown that maintenance PI/r monotherapy, in HIV-1-infected individuals who are already fully suppressed with standard ART, is able to maintain viral load suppression for months in most individuals. However, viral rebound rates in the long term are higher in patients receiving PI/r monotherapy than triple ART, 16 and thus it is not currently recommended in US and European treatment guidelines. 17,18 Although residual plasma viraemia below the detection limit of standard assays (,50 HIV-1 RNA copies/mL) has been suggested to be the origin of viral failures in this context, there is no clear evidence available. 19,20 In the present single-arm pilot study, we evaluated the effect of intensification with raltegravir on HIV-1-infected individuals on maintenance PI/r monotherapy by measuring changes in residual viraemia, low-level viral replication, HIV-1 DNA reservoirs, T cell immune activation and soluble inflammatory biomarkers.
Although median values of residual viraemia showed no major changes during the study, we found that the probability of having intermediate levels of residual viraemia (10-60 copies/mL) was significantly higher in the pre-and post-raltegravir intensification periods than during the 24-week intensification period, suggesting an impact of treatment intensification on this parameter. This is in contrast with the absence of reductions in residual plasma viraemia during raltegravir intensification in triple therapies. 7,21,22 Thus, our results suggest the lack of complete viral suppression that might explain the lower efficacy rates observed in PI/r monotherapies compared with standard ART.

Puertas et al.
Of note, we systematically detected 2-LTR circles in those patients even before the addition of raltegravir to the PI/r regimen. As 2-LTRs are surrogate markers of recent infection events, 23 these results indicate the persistence of viral replication. Furthermore, a transient increase was observed after raltegravir initiation, as reflected by the accumulation of 2-LTR peaks between weeks 1 and 8. Taken together, our data demonstrate the persistence of low-level viral replication in a significant proportion of individuals on suppressive PI/r monotherapy. It is also possible that active replication occurs in anatomic compartments that are less accessible to PIs. 9 Still, we did not find any correlation between the detection of 2-LTR circles and the presence of intermediate residual viraemia levels in the patients before raltegravir intensification and neither of those parameters were preferentially associated with darunavir or lopinavir-based regimens.
In accordance with data from studies comparing monotherapy and triple therapy, 24,25 this low-level viral replication observed in PI/r-treated individuals does not seem to drive significant replenishment of the proviral reservoir in the short term, as a 24-week intensification period did not impact on levels of cell-associated viral DNA, at least in circulating peripheral CD4! T cells.
In contrast with the results from previous intensification studies, 10,11 changes in CD8! T cell activation were modest in the present study. Indeed, no changes were observed in the frequency of CD38! HLA-DR! CD8! T cells, although a significant reduction in CD38 expression was observed in the whole CD8! T cell population throughout the intensification period. This change was unrelated to changes in the frequency of naive cells and confirms larger reductions of CD38 expression compared with HLA-DR expression in intensification strategies. 26 However, in this case, CD38 changes after raltegravir intensification were close to the natural variation of CD38 expression during non-intensification periods. When a post hoc analysis was performed after classifying participants according to the detection of 2-LTR circles, we observed the previously reported higher CD8! T cell activation in 2-LTR! individuals; 7 however, no specific or significant decrease of activation was induced by intensification with raltegravir in those participants. In summary, we found an unanticipated minor effect of raltegravir on CD8! T cell immune activation.
Similarly, immunological follow-up revealed no major changes in the frequency of CD4! or CD8! subsets during the intensification period. However, a striking transient increase in CTLA-4 was noticed in both CD4! and CD8! T cells after intensification with raltegravir. The increase peaked between weeks 4 and 8 and was unrelated to the presence of detectable levels of 2-LTRs or any other virological or immunological parameter analysed. CTLA-4 is a negative regulator of T cell receptor-mediated CD4! T cell activation and function, which is upregulated on HIV-specific T cells; 27,28 therefore, suppression of HIV replication with ART is associated with decreased expression of CTLA-4 and other inhibitory receptors, such as PD-1 and TIM-3, on HIV-specific CD4! T cells. 27,[29][30][31][32] Moreover, CTLA-4 is reported to be moderately overexpressed in the total CD4! population during HIV infection and its expression correlates inversely with CD4! T cell count and positively with immune dysfunction and exhaustion, which are associated with a progressive loss of effector function and disease progression. 27,28 Consistent with these findings, we observed the highest frequency of CTLA-4! cells within the effector memory CD4! T cell subset (data not shown) and a trend toward higher CTLA-4 expression in individuals with detectable levels of 2-LTR circles. The meaning of these transient changes in CTLA-4 expression that were observed in all memory subsets remains unknown and warrants further exploration.
Recent studies have reported that factors associated with virological rebound in individuals receiving PI/r monotherapy include low nadir/baseline CD4! T cell counts and shorter duration of viral suppression. 33,34 In this sense, the restrictive inclusion criteria applied in the present study ensured that the individuals had a favourable virological and immunological status. Indeed, the participating individuals were able to remain on PI/r monotherapy for a median of 3.3 years, again suggesting a better long-term viral control than those who had to return to ART. However, although the participants' characteristics might have been an unexpected limiting factor in the present study, our data indicate that, at least in long-term suppressed individuals with a good CD4! T cell count at baseline, PI/r monotherapy can restrain immune activation.
Overall, temporary raltegravir addition uncovered low-level viral replication in a significant proportion of individuals on PI/r monotherapy, as evidenced by the transient increase of viral 2-LTRs circles in peripheral CD4! T cells. Furthermore, intermediate residual viraemia (10-60 HIV-1 RNA copies/mL) detected during PI/r monotherapy became less frequent during raltegravir intensification. Those results evidenced the incomplete viral suppression occurring in patients on long-term PI/r monotherapy, thus being the potential origin of the lower viral suppression efficacy of this therapeutic simplification strategy, and suggest that dual therapy of PI/r and integrase inhibitors might improve virological parameters. contributed to data analysis and interpretation, and drafted the manuscript including figures and tables; all authors revised it critically for important intellectual content and have approved the final version submitted for publication. All authors fulfil ICMJE criteria for authorship, have had access to all the data in the study and agree to be accountable for the work in ensuring that questions related to its accuracy or integrity are appropriately investigated and resolved.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Psychology

text:
Epigenetic dysregulation of Oxtr in Tet1- deficient mice has implications for neuropsychiatric disorders

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice — by deleting the largest coding exon 4 (Tet1Δe4) — we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4–/– mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4–/– mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders. Research Article Genetics Neuroscience

Introduction
Oxytocin, the brain's most abundant neuropeptide, acts as a neuromodulator and hormone to its G protein-coupled oxytocin receptor (OXTR) and modulates a variety of behaviors including social memory and recognition, sexual and aggressive behaviors, and bonding and maternal care (1)(2)(3)(4). OXTR is a small gene, with only 4 exons, and the coding exon spans only a portion of exons 3 and 4 (5,6). The expression of OXTR in mammals is cell type specific and developmentally regulated (7,8). The expression of OXTR is higher during parturition and lactation in the uterus and mammary tissues, respectively, as well as in the brain during mating, social bonding, and child rearing (2). The mechanism underlying this dynamic transcriptional regulation is poorly understood. The involvement of DNA methylation in regulating the expression of OXTR has been suggested, but the exact mechanism mediating this process remains largely unknown (9)(10)(11). Epigenetic studies in humans have found altered DNA methylation in the 5′ CpG island of OXTR in a wide spectrum of morbid behaviors and neuropsychiatric disorders, such as autism spectrum disorder (ASD), schizophrenia, bipolar, depression, obsessive-compulsive disorders (OCD), and anxiety (12)(13)(14)(15)(16)(17)(18). Similar correlations between DNA methylation and behavior are also reported in other species, such as dog and nonhuman primates (19,20).
The discovery of the ten-eleven translocation (TET) family of methylcytosine dioxygenases has shed light on the regulation of DNA methylation during the development (21,22). TET proteins catalyze active demethylation of DNA in a stepwise process through formation of 5-hydroxymethylcytosine (5hmC) and OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice -by deleting the largest coding exon 4 (Tet1 Δe4 ) -we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1 Δe4-/mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1 Δe4-/mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.
other oxidative derivatives of 5-methylcytosine (5mC) (23,24). The mouse Tet gene family (Tet1-3) is differentially regulated during development and in adult brain (25). Tet1 expression is relatively enriched in embryonic stem cells (ESCs) (25,26) and has been implicated in regulating genes important for cellular differentiation, pluripotency, and neurogenesis (27)(28)(29). In addition to being a DNA demethylation intermediate, 5hmC is a stable DNA modification and is present at variable levels in ESCs and adult tissues, but it is specifically enriched in postnatal brains (30)(31)(32), where it accumulates with age (33). The primary role of 5hmC and TET protein is believed to contribute to transcriptional regulation of the genes that are important for the brain development and function (34)(35)(36)(37)(38)(39)(40)(41). However, the exact molecular mechanism underlying these processes has not been completely elucidated. One hypothesis for brain enrichment of 5hmC is that key neural genes important for brain function are regulated by TET-mediated hydroxymethylation. Identification of such genes would be the first critical step to investigate the function of TET proteins and 5hmC in the brain.
Three lines of Tet1 mutant mice that disrupt different portions of coding exons have been reported (42)(43)(44). Studies of these mutant mice have identified several neural genes that are dysregulated in Tet1-deficient mouse brains (42,43). However, the findings from these studies of Tet1 mutant mice reveal some similarities, as well as significant differences. The reasons responsible for these differences are not immediately clear. A recent report of different Tet1 isoforms due to the alternative splicing during the early development may suggest that mutations targeting different exons could have different molecular consequence (37,42,45,46).
From the generation and analysis of a new line of Tet1 mutant mice, we discovered that the expression of the Oxtr gene was significantly reduced in the brain of TET1-deficient mice. Unexpectedly, we uncovered that Oxtr displays a complex transcriptional regulation and an array of mRNA isoforms in the brain. The reduced expression of Oxtr only affected select mRNA isoforms in Tet1-deficient brains. Accordingly, the CG sites within the 5′ CpG island of Oxtr was selectively hypermethylated in embryonic and postnatal brains. Thus, we provide the first evidence to our knowledge of an epigenetic mechanism capable of directly regulating Oxtr expression in vivo. These discoveries provide a molecular framework for reinterpreting findings from existing genetic and epigenetic studies and, more importantly, are valuable to guide the experimental design of future studies of the transcriptional regulation and the role of OXTR in neuropsychiatric disorders.

Results
Generation of a line of Tet1 mutant mice and homozygous ESCs. Previous studies have reported 3 lines of Tet1 mutant mice that delete coding exon 5, exons 11-13, or by inserting a gene trap (Gt) in intron 2 (44) (Figure 1A). We generated a new line of Tet1 mutant mice by targeting and floxing the largest coding exon, exon 4 ( Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.120592DS1). Deletion of exon 4 causes a frameshift, leading to a premature stop codon in exon 6 that is 36 amino acids after the end of exon 3 and results in disruption of the catalytic domain of TET1. Expression analysis by reverse transcription PCR (RT-PCR) confirmed exon 4 deletion ( Figure 1B, right panel), and Western blot analysis showed deficiency of TET1 protein from Tet1 Δe4-/mouse hippocampus ( Figure 1C). TET1 has been suggested to promote transcription of pluripotency factors by an in vitro study using RNAi and plays a distinct role during the different stages of the development (47,48). Therefore, we generated homozygous Tet1 Δe4-/-ESCs by targeting the second allele of Tet1 e4f ESCs sequentially, followed by introduction of Cre in Tet1 e4f/f ESCs ( Figure 1D and Supplemental Figure 1, B-E). Tet1 Δe4-/-ESCs are viable with no apparent abnormality in cellular morphology. Tet1 and Tet2, but not Tet3, are known to be highly expressed in ESCs (25,26). Accordingly, we found Tet2 -but not Tet3 -upregulated in Tet1 Δe4-/-ESCs ( Figure 1E), suggesting a compensatory mechanism of Tet2 in response to deficiency of Tet1 in ESCs. However, this similar compensatory mechanism was not observed in adult hippocampus ( Figure 1F). Tet1 Δe4+/mice were backcrossed to C57BL/6J mice for more than 6 generations (N6) before molecular experiments and behavioral analysis. Tet1 Δe4+/mice on a C57BL/6J background displayed partial perinatal lethality and a deviation from th expected Mendelian ratio of genotypes at weaning age, indicating a role for TET1 in early development ( Table 1). The surviving Tet1 Δe4-/mice showed a mild degree of growth retardation, as Tet1 Δe4-/mice weighed significantly less than Tet1 +/+ littermates at weaning; this persisted into adulthood ( Figure 1G). Tet1 Δe4-/mice did not have any apparent abnormal behavior in their home cage.
We hypothesized that TET1-mediated accumulation of 5hmC is implicated in neuronal activity-regulated gene transcription in the brain. Tet1 is expressed quite ubiquitously in the postnatal human and mouse brains with relative higher expression in the hippocampus (Supplemental Figure 2, A and B). Because neuronal activity-regulated gene transcription has been studied extensively in hippocampus (50), we then focused our studies in the hippocampus of Tet1 Δe4-/mice using an electroconvulsive stimulation (ECS) paradigm to induce neuronal activity. We first validated the activity-dependent expression paradigm by confirming upregulation of Bdnf and Homer1a after ECS (Supplemental Figure 3A) (51,52). The effect of TET1 deletion on activity-dependent gene regulation was examined by comparing RNA sequencing (RNA-seq) expression profiles from the hippocampus of ECS-treated Tet1 Δe4-/and Tet1 +/+ mice (Supplemental Figure 3B). The sequence coverage and depth are summarized in Supplemental Figure 3B. Examination of RNA-seq data reads mapped to Tet1 confirmed the deletion of exon 4 (Supplemental Figure  3C). Using a FDR < 0.05 to compare the genes differentially expressed between Tet1 Δe4-/and Tet1 +/+ mice, we discovered that 34 genes were significantly upregulated and 184 genes were downregulated ( Figure 2A and Supplemental Table 1), suggesting a primary role for TET1 in activating gene expression. In order to test the hypothesis that TET1 is responsible for regulating activity-dependent genes, we cataloged a dataset of activity-dependent genes based on a literature review (Supplemental Table 2). While there was no significant enrichment for neuronal activity-regulated genes disrupted in Tet1 Δe4-/mice in the RNA-seq data, dysregulation of several activity-dependent genes was identified, including the master memory gene regulator Npas4 ( Figure 2B) (53). Npas4 downregulation was confirmed by quantitative PCR (qPCR) in the hippocampus of untreated Tet1 Δe4-/mice, as well ( Figure 2C). A similar finding was previously reported in another line of Tet1 mutant mice (37). Gene Ontology (GO) analyses by DAVID of the dysregulated genes detected a significant enrichment for genes involved in the extracellular region/matrix/space ( Figure  2D). In examining and verifying other nonactivity-dependent genes in RNA-seq data, we found significant downregulation of Oxtr, the transmembrane receptor that binds oxytocin and a key neuronal gene implicated in brain function and behavior. Oxtr downregulation was also observed in untreated Tet1 Δe4-/mice but not in Tet1 Δe4-/-ESCs ( Figure 2E).
We compared our list of Tet1 Δe4-/dysregulated genes from ECS-treated hippocampus to the dysregulated genes reported in Tet1 exon 5-deficient (Δe5) hippocampus (37,43) and Tet1 exon 11-13 deletion (Δe11-13) neural progenitor cells (NPCs) (42). In Tet1 Δe5 mutant mice reported by Rudenko et al., a total of 258 genes are dysregulated in the hippocampus (37). A significant set of genes (54 genes, 20.9%) was also found to be altered in the hippocampus of Tet1 Δe4-/mice ( Figure 2F). In NPCs of Tet1 Δe11-13 mice, only 4 of 54 genes (7.4%) overlapped with the dysregulated genes in Tet1 Δe4-/mice. When we compared dysregulated genes from Tet1-knockdown ESCs by RNAi (47) or Tet1 Gt primordial germ cells (44), we did not find significant overlap with the dysregulated genes in the hippocampus of Tet1 Δe4-/mice. These comparisons indicate TET1 differentially regulates sets of genes in a tissue-dependent manner. The differences between Tet1 mutant mice may be due to the nature of the mutations disrupting specific isoforms of Tet1, as suggested by the alternative splicing of the Tet1 gene in a recent report (45).
DNA hypermethylation of the CpG island of Npas4 and Oxtr in Tet1 Δe4-/mice. Based on the role of TET1 in DNA demethylation, we examined if the downregulation of Npas4 and Oxtr were associated with increased CpG island DNA methylation. We used targeted bisulfite genomic sequencing to examine the CpG island associated with the Npas4 promoter region and found it significantly hypermethylated in Tet1 Δe4-/hippocampus (27.2% ± 3.1%) compared with Tet1 +/+ (8.1% ± 0.6%) ( Figure 3A and Supplemental Figure 4A). This is in line with previous data supporting a role for TET1 in regulating the methylation state of Npas4 (37).
The mouse Oxtr gene contains 4 exons (6) including a 5′ UTR, which encompasses exons 1, 2, and part of exon 3 and overlaps with an 859 bp CpG island ( Figure 3B). The human CpG island spans 2,319 bp, Genotype ratio at weaning. Fewer Tet1 Δe4-/mice survived to weaning (χ 2 (2, n = 248) = 11.903, P < 0.001). extending to the more 5′ noncoding region of OXTR (see detail in UCSC genome browser for both mouse and human OXTR/Oxtr CpG islands; www.genome.ucsc.edu). The mouse Oxtr gene also has high GC-content in the homologous region to the human promoter CpG island, indicating that the difference in CpG island length is likely due to a difference in computational thresholds rather than a biological difference. The CpG islands in both human and mice are located downstream of the characterized canonical promoter (5, 6) ( Figure 3B). This suggests the presence of additional regulatory elements, such as an additional promoter within the CpG island. In silico analysis of ENCODE data also revealed histone modification peaks within the CpG island that support the presence of regulator elements for OXTR. To examine if the reduced hippocampal expression of Oxtr was associated with DNA hypermethylation of potential regulatory elements within the Oxtr gene, methylation in the 5′CpG island and adjacent region (BS1-BS3) of hippocampus of adult mice was analyzed by targeted bisulfite sequencing ( Figure 3B). We first performed bisulfite sequencing on BS1, a region mostly upstream of the 5′ Oxtr transcription start site (TSS). Overall, we were able to achieve >99.0% of the C-T bisulfite conversion rate for most experiments, and only the sequence products that reached >99% C-T conversion rate were included for data analysis. Consistent with the association of low DNA methylation at transcriptionally active promoter regions, BS1 is mostly unmethylated and comparable between Tet1 +/+ (8.45% ± 0.51%) and Tet1 Δe4-/-(10.13% ± 0.66%) mice, indicating that methylation in the BS1 region is probably not responsible for the reduced expression of Oxtr ( Figure 3, B and D, and Supplemental Figure 4B). We next examined BS2, a region just downstream of the most 5′ TSS. A portion of this region has been shown in humans to have activity for an alternative promoter that is suppressed by DNA methylation (region MT2 in Figure 3B) (9). Tet1 Δe4-/mice have significantly increased DNA methylation (9.55% ± 1.48%) in BS2 compared with their Tet1 +/+ littermates (2.62% ± 0.61%) (P = 0.0015) ( Figure 3, B and D, and Supplemental Figure 4B). The differential methylation in the BS2 region appears to be driven by only a few CpG dinucleotides, including the CpG sites that may be homologous to the human CpG -934 (The "A" in the ATG codon is denoted as +1 position) which was hypermethylated in a small sample of autistic individuals (14) and correlates with several morbid behaviors (54)(55)(56). ChIP data from the ENCODE project (57) indicate that the CpG island spanning Oxtr exon 3 includes a potential regulatory element. Strikingly, nearly every CpG assessed in this region (BS3) is significantly hypermethylated in Tet1 Δe4-/mice (70.6% ± 3.60%) compared with their Tet1 +/+ littermates (22.03% ± 3.23%) (p<0.0001) ( Figure 3, B and D, and Supplemental Figure 4B). To test for a gene dosage effect of TET1 on Oxtr DNA methylation, we examined the BS3 region in heterozygous Tet1 Δe4+/mice and found an intermediate level of methylation (42% ± 3.3%) that is significantly lower than Tet1 Δe4-/-(n = 4, P = 0.0019) ( Figure 3, B and D, and Supplemental Figure 4B). Because bisulfite genomic sequencing method cannot distinguish between 5mC and 5hmC, we performed hydroxymethylated DNA immunoprecipitations (hMeDIP) using an antibody specific for 5hmC for the BS3 region, and we conducted qPCR on immunoprecipitated DNA. This analysis did not reveal any dignificant difference in 5hmC abundance in the BS3 region between Tet1 -/and Tet1 +/+ animals. This result further supports that the difference in methylation detected by bisulfite sequencing is due to a difference in 5mC, not 5hmC ( Figure 3C). We examined the level of BS3 methylation in ESCs to test if the correlation between Oxtr gene expression and DNA methylation extended to ESCs. Consistent with our findings that Oxtr was not differentially expressed in ESCs, BS3 methylation in Tet1 Δe4-/-ESCs is not different from Tet1 +/+ ESCs ( Figure 3E). These data suggest that DNA methylation of BS3 regions is associated with altered Oxtr expression, as ESCs do not express Oxtr. To examine brain region specificity of Oxtr CpG island hypermethylation, we examined multiple brain regions and found the hypermethylation of BS2 and BS3 in Tet1 Δe4-/adult mouse brain is not limited to hippocampus but also includes the regions of cerebellum, cerebral cortex, and olfactory bulb ( Figure 3F and Supplemental Figure  4C). These results suggest that hypermenthylation of Oxtr in Tet1 Δe4-/animals may be established early in brain development prior to the development of different brain regions. We examined BS3 in embryonic brains and found hypermethylation of Oxtr in the cerebrum as early as E14.5 ( Figure 3G). As it is technically challenging to genotype and analyze the DNA methylation patterns of each germ layer of an individual embryo due to an insufficient amount of DNA, we speculated that if hypermethylation of BS3 was established and maintained prior to gastrulation, we would find adult tissues from all 3 primary germ layers to be hypermethylated. In addition to our finding of hypermethylation in the adult brain (ectoderm), we observed BS3 hypermethylation in tissue that arose from mesoderm (heart) and endoderm (lung) in adult Tet1 Δe4-/mice ( Figure 3H). As BS3 methylation is similar in ESCs between WT and Tet1 Δe4-/-, these data suggest a window during early embryonic development for TET1-mediated maintenance of Oxtr methylation, which is TET1-dosage dependent. The TET1 protein contains a CXXC domain, which preferentially binds to unmethylated CpG-rich sequences (58). We performed whole-genome bisulfite sequencing in adult cerebral cortex tissue using a previously described method (45,59) to investigate the genome-wide effect of TET1 deficiency on DNA methylation in the brain. As whole genome bisulfite sequencing requires larger quantities of input DNA, cerebral cortex tissue was chosen because the size of the tissue allows for a sufficient amount of DNA to be obtained from a single animal. Sequence reads were mapped to mouse genome mm9 assembly (http:// genome.ucsc.edu) using BSMAP program (60), and only samples with C to T conversion rate greater than 99% were accepted for further data analysis. Through this analysis, we identified 666 differentially methylated regions (Tet1-DMRs) between Tet1 Δe4-/and Tet1 +/+ , with an enrichment for hypermethylated Tet1-DMRs (522 hypermethylated vs. 144 hypomethylated). We discovered an enrichment of hypermethylated Tet1-DMRs in CpG islands (380 CpG islands; ΔmCG ≥ 20%), including the Oxtr CpG island (1.2 fold observed/random, P = 1.25 × 10 -7 ) (Supplemental Table 3). The distributions for DMRs indicated a distinct pattern across the genome. Stratifying CpG islands by location (promoter vs intragenic) revealed Tet1-DMRs significantly enriched in intragenic CpG islands ( Figure 3I, 4.5-fold observed/random, P = 2.65 × 10 -38 ), indicating a more prominent role for TET1 in regulating intragenic CpG island methylation. The motif analysis by HOMER2 program has revealed top 20 binding sites including NeuroD and MEF2C, which are known to be important for transcriptional regulation of neuronal genes (46) (Supplemental Figure 5B). In addition, the methylation at the mCH site is not significantly different between Tet1 +/+ and Tet1 Δe4-/mice (Supplemental Figure 5A).

Discovery of Oxtr gene mRNA isoforms and reduced expression of Oxtr Isoforms in Tet1
Δe4-/mice. The pattern of Oxtr hypermethylation within BS3 and not BS1 suggests that a regulatory element lies within exon 3 of Oxtr, which could cause Oxtr to have a more complex transcript structure than described in the literature (5,6,61). A single Oxtr transcript (Oxtr-A) has been previously characterized with an ORF spanning exons 3 and 4 and encodes a 388-amino acid protein (6) ( Figure 4A). Examining the Oxtr gene structure in the genome browser (mm9, http://genome.ucsc.edu/) revealed a predicted mRNA isoform lacking exons 1 and 2 (Oxtr-B), as well as a previously deposited isoform originating from intron 3 (Oxtr-C). We validated the presence of all 3 isoforms in the hippocampus by RT-PCR and sequencing ( Figure 4A). In addition, we identified a fourth isoform by RT-PCR and sequencing in which exon 3 is skipped (Oxtr-D). To examine if there were additional TSS not previously reported that could be affected by the exonic hypermethylation of the BS3 region, we performed 5′ rapid amplification of cDNA ends (RACE) using primers from exon 4 in a pair of Tet1 +/+ and Tet1 Δe4-/mice. Interestingly, the sequences of 5′ RACE products revealed 4 potentially novel TSS, including 1 originating from intron 3 (Oxtr E-H) ( Figure 4A). Isoforms A and B have the same full-length ORF, while the other mRNA isoforms have predicted shorter ORFs ranging from 75-114 amino acids. However, whether the shorter mRNA isoforms are translated endogenously and possess a function similar to OXTR warrants further investigation. We measured the relative abundance of these various Oxtr transcripts by qPCR with primer sets indicated in Figure 4A. Our results indicate that Oxtr-B is the most dramatically reduced isoform (20% of WT), while full-length Oxtr-A and potentially novel isoform Oxtr-H are not different in Tet1 Δe4-/hippocampus ( Figure 4B). Transcript levels of isoforms C and D were below our detection threshold, and unique primers to detect isoforms E, F, and G could not be designed because of transcript overlap. Our data together indicate a complex transcriptional regulation with the possibility of more than 1 TSS of Oxtr. This data also suggests a mechanism of TET1-governed DNA methylation underlying expression of selective isoforms of Oxtr.
The histone modifications are altered in DMRs of Oxtr in Tet1 Δe4-/mice. DNA methylation is mechanistically known to be linked to various histone modifications in the genome (62). DNA hypermethylation of BS3 and reduced transcripts of Oxtr in Tet1 Δe4-/mice prompted us to examine whether histone modifications associated with Oxtr are altered. We first examined the ChIP-seq data of mouse brain in the Oxtr region from the ENCODE project (http://genome.ucsc.edu) (63). Consistent accumulation of both the active histone mark H3K4me3 and the repressive mark H3K27me3 suggest that a bivalent peak of histone modifications may also contribute to expression of Oxtr in various tissues ( Figure 4C and Supplemental Figure 6). For example, the peaks of H3K4me3 ChIP-seq data in heart tissue show a distinct pattern at E14.5 and 8-week-old mice during development (Supplemental Figure 6). The bivalent chromatin regions overlap with the canonical TSS and the hypermethylated BS3/BS2 region within CpG island in Tet1 mutant mice. These in silico data strongly suggest additional transcriptional regulatory elements in BS3/BS2 hypermethylated that are associated with different histone modifications. Oxtr in mouse brain. The enrichment of histone H3K4me3 and H3K27me3 in BS1-BS3 regions of Oxtr was revealed from ENCODE project. A second putative regulatory element was identified within exon 3 (large black bar) of Oxtr, which overlaps a CpG island (green bar) (mm9, http://genome.ucsc.edu/). Selection of Oxtr regions for bisulfite sequencing (BS1-BS3) and ChIP-PCR are indicated. ENCODE ChIP data shown is from E14.5 whole brain produced in the laboratory of Bing Ren (H3K4me3, GEO GSM1000095; H3K27me3, GEO GSM1000143). The similar pattern is also observed in ChIP-seq from 8-week cerebellum We then compared the histone modifications of H3K4me3, H3K27me3, and H3K4me1 between the DMRs identified in the cerebral cortex of Tet1 Δe4-/mice and randomly selected 2 kb bin control genomic regions genome-wide from the same methylome using ChIP-seq data in mouse cortex deposited by ENCODE (63). This analysis revealed that the modifications of H3K4me3, H3K27me3, and H3K4me1 were enriched in the DMRs of Tet1 Δe4-/mice compared with the control regions ( Figure  4D). Accordingly, ENCODE ChIP-seq data from E14.5 whole brain tissue indicates the Oxtr promoter has 2 active H3K4me3 peaks and a repressive H3K27me3 peak that overlap the BS1-3 regions ( Figure  4C), typical of a bivalent domain (64). TET1 is known to preferentially bind to these transcriptionally poised bivalent domains, which are generally hypomethylated in ESCs (47). To examine whether the histone modifications of H3K4me3 and H3K27me3 suggested from in silico analysis were altered in Tet1 Δe4-/mice, we performed ChIP analysis using hippocampus tissues and GADPH as control to show the specificity of the antibody ( Figure 4E). H3K4me3 was increased and H3K27me3 was not changed in the BS1 region ( Figure 4F). H3K4me3 and H3K27me3 were reduced in the hypermethylated BS3/ exon 3 region in Tet1 Δe4-/mice ( Figure 4G). The degree of reduction of H3K4me3 (active chromatin mark) was more prominent than that of H3K27me3 (repressive chromatin mark). Hypermethylation of Oxtr skews the balance between the active and repressive marks, resulting in abundance of H3K27me3 and the reduction of select mRNA isoforms of Oxtr.
Tet1 Δe4-/mice display abnormal social behaviors. Due to the observed dysregulation of Npas4 and Oxtr and their roles in synaptic development and function (65,66), we wanted to test whether there would be cognitive and social deficits in Tet1 Δe4-/mice. We performed a battery of behavioral tests using multiple cohorts of backcrossed (N6) C57BL/6J mice (Supplemental Figure 7). Tet1 Δe4-/mice appeared grossly normal by a neurophysiological screen (Supplemental Figure 8). Tet1 Δe4-/mice were hypoactive and spent less time in the center of the open field ( Figure 5A and Supplemental Figure 9A) but showed an enhanced ability to stay on the accelerating rotarod (Supplemental Figure 9B), suggesting the hypoactivity in the open field is more likely anxiety related than a motor impairment. However, Tet1 Δe4-/mice do not have a strong anxiety phenotype in the light-dark emergence test (Supplemental Figure 9C).
The role of OXTR has been well studied in the context of social and maternal care behaviors (66, 67), and increased aggression and reduced maternal care are observed in Oxtr mutant mice (68). To examine whether Tet1 Δe4-/mice show similar abnormal behaviors, we first performed the resident intruder test. In the sex-matched resident intruder test, there was a significant increase in agonistic behaviors in female Tet1 Δe4-/mice but not in male Tet1 Δe4-/mice ( Figure 5B and Supplemental Figure 9D). Tet1 Δe4-/female mice display threatening postures of a similar magnitude to male mice. These abnormal behaviors are similar to that observed in Oxtr -/mice (68). We further evaluated the maternal care behavior by virgin pup-retrieval test.
Maternal care behavior was only tested in virgin females, as Tet1 Δe4-/female mice have reduced fertility and could not produce enough pups for dam pup retrieval. Increased maternal responsiveness after repeated exposures to pups is associated with increase oxytocin expression in the brain of WT virgin female mice (69,70). Interestingly, the virgin pup retrieval test revealed that Tet1 Δe4-/female mice show increased latency to retrieve pups on the third day of testing ( Figure 5C, left panel), spend less time crouching over pups ( Figure  5C, middle panel), and show an increase in aggressive interactions of pup exposure ( Figure 5C, right panel). Male mice were not tested in virgin pup retrieval, as males are known to attack nonoffspring (71).
In the potentially novel object recognition test, Tet1 Δe4-/mice show a trend toward short-term (1 hour) memory deficits, indicating impairment in episodic memory ( Figure 5D). Long-term (24 hours) memory is not significantly different between genotypes. Spatial learning (Morris water maze) (Supplemental Figure 9, E and F), olfactory memory (social transmission of food preference) (Supplemental Figure 9G), and associative memory (fear conditioning) did not differ between Tet1 Δe4-/mice and their Tet1 +/+ littermates (Supplemental Figure 9H).
(http://genome.ucsc.edu) (Supplemental Figure 6). (D) The enrichment of H3K4me3, H3K27me3, and H3K4me1 in DMRs of Tet1 Δe4-/mice. The hypermethylated DMRs (200 bp bin) in Tet1 Δe4-/cortex was selected firstly by Fisher test with a threshold of P < 0.05. The 2000 bp genomic windows containing at least 4 hypermethylated DMRs (200 bp) in Tet1 Δe4-/cortex were selected out as DMRs for histone enrichment analysis. Random control regions with same bin size covered by the same methylome were chosen as controls. The ENCODE ChIP-seq data from WT adult mouse neocortex was used for the analysis. P values (calculated by t test) indicate a significant differential histone enrichment between DMRs and control region. (E-G) ChIP-qPCR revealed altered histone modifications at the bivalent promoter region of and GARDP control (E) and Oxtr (F and G) in the cerebrum of Tet1 Δe4-/mice. H3K4me3 (active mark), H3K27me3 (repressive mark), and IgG (isotype negative control) were assessed in 2 regions overlapping coding exon 3 and exon 1 in the 5′ UTR. Both H3K4me4 and H3K27me3 were reduced in BS3 hypermethylated region of Tet1 Δe4-/mice (n = 2/group and 3 replicates for each group).
Tet1 Δe4-/mice display impaired response to OXTR agonist stimulation but normal synaptic plasticity in the hippocampus. The reduced expression of Oxtr and the similar behavioral phenotype of Tet1 Δe4-/to Oxtr -/mice prompted us to examine the functional deficits specific to Oxtr. It has been shown that OXTR agonist (TGOT) increases the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) onto hippocampal CA1 pyramidal neurons (72,73). We speculated that if the reduced expression of Oxtr in Tet1 Δe4-/neurons affected the function of OXTR, the response to oxytocin in pyramidal cells could be impaired. Thus, we recorded sIPSC from CA1 pyramidal cells at baseline, as well as in the presence of TGOT stimulation. The mean frequency at baseline for Tet1 Δe4-/-(4.16 ± 0.67 Hz) is borderline lower than that of Tet1 +/+ (2.47 ± 0.48 Hz) ( Figure 6A). The increase of sIPSC frequency in response to TGOT in Tet1 Δe4-/is similar to that of Tet1 +/+ mice. The amplitude of sIPSC of Tet1 Δe4-/neurons (26.56 ± 6.78 pA) is comparable with that of Tet1 +/+ mice (20.36 ±2.19 pA). However, the amplitude of sIPSC in response to TGOT in Tet1 -/neurons is significantly diminished compared with that of Tet1 +/+ ( Figure 6B). A previous study reports the impaired long-term synaptic plasticity in hippocampus of mutant mice lacking both DNA methyltransferases Dnmt1 and Dnmt3a in postnatal brain, suggesting that DNA methylation is essential for synaptic function (74). We then examined whether deficiency of Tet1 may also affect synaptic function. The baseline synaptic transmission ( Figure 6C) and the presynaptic function measured by paired pulse facilitation (PPF) and fiber volley are not affected (Figure 6, D and E). In contrast to the impaired longterm potentiation (LTP) in the hippocampus of Dnmt1 and Dnmt3a mutant mice, the synaptic plasticity as measured by hippocampal LTP in hippocampal CA synapses were not altered in Tet1 Δe4-/mice ( Figure 6F).

Discussion
OXTR, the receptor for oxytocin, is a 9-amino acid neuropeptide produced in the brain and functions in both peripheral reproductive tissues and in the brain (2, 3). The molecular mechanism regulating the dynamic expression of OXTR in response to different physiological stimuli has not been characterized. Our discovery of the complex transcriptional regulation and hypermethylation of Oxtr in Tet1 Δe4-/mice provide insight into transcriptional regulation of Oxtr and support that Oxtr is epigenetically regulated and that hypermethylation of CpG island correlates with the reduced expression of Oxtr isoform in vivo.
Previous in vitro studies have suggested that 2 regions may implicate DNA methylation in expression of Oxtr. First, the methylation of 2 CpG sites (-932 and -733; the canonical TSS within the exon 1 is numbered as +1) in a region overlapping with the BS1 analyzed in this study has been shown to be negatively correlated with the expression of Oxtr in the mouse 4T1 cell line and brains (10,11). Second, the methylation of CpG site in a 450 bp region (MT2) downstream of 5′ canonical TSS that overlaps the homologous BS2 region is important for decreased expression of human OXTR in vitro (9). In brain regions analyzed in Tet1 Δe4-/mice, we did not find a significant methylation difference for BS1, but we did discover a significant difference in the BS2 and BS3 regions. Consistent with these results, the full-length isoform Oxtr-A, for which the transcript begins in the BS1 region, was unaltered in Tet1 Δe4-/mice. Isoform Oxtr-B, a short isoform initiated from an alternative downstream promoter in BS2-BS3 region appeared most affected by the hypermethylation.
The human -934 (The A in the ATG codon is denoted as +1 position) and adjacent CG sites, regions that have been studied in many epigenetic-associated studies in human psychiatric disorders, are homologous to the hypermethylated region in the BS2 in Tet1 Δe4-/mice based on the in silico analysis of genomic position. However, it is difficult to pinpoint the exact CG site in mice that is equivalent to the human CpG -934 CG site. The discovery of potentially novel mRNA isoforms in our study presents many questions about the transcriptional regulation of Oxtr. It remains to be determined whether each mRNA isoform has brain region-or cell type-specific expression or if each isoform is translated into a protein and how the methylation state of individual CG sites regulate isoform-specific Oxtr expression. Nevertheless, our finding raises interesting questions about whether each isoform may possess a specific function in the developing or adult brain.
Our Tet1-KO mouse with a deletion of exon 4 is potentially novel. While we recognize the reports of other 3 different lines of Tet1 mutant mice (42)(43)(44), we believe that our line of Tet1 mutant mouse still has significant value for future study of TET1 function in vivo because of possible alternative splicing of Tet1 suggested by a recent report (45) and in silico data. The dysregulation of Npas4 is consistent with the finding from the Tet1 Δe5 line of Tet1 mutant mice (37). The reduced expression of Oxtr in Tet1 Δe4-/mice is potentially novel and has not been addressed in other lines of Tet1 mutant mice. Our study suggests that early prenatal development is a critical period for TET1 function, as was also reported in a previous in vitro study (29) and a recent report indicating a lineage-specific role of TET1 in embryonic development (75). The Oxtr CpG island is hypermethylated in the E14.5 cerebrum, as well as mesodermal and endodermal tissues, but not in ESCs. The most plausible explanation would be that, without TET1, the Oxtr CpG island becomes hypermethylated during the onset of de novo methylation between E4.5-7.5 and the aberrant methylation is then propagated in every cell from that point. Rather than a role in active demethylation, the hypermethylation of Oxtr in Tet1 mutant mice supports a role for TET1 in preventing de novo methylation, as has previously been suggested (29,76). In addition, the finding of intermediate hypermethylation of Oxtr in heterozygous Tet1 Δe4+/mice suggests that proper establishment of DNA methylation during development is sensitive to levels of TET1.
The role of oxytocin and OXTR in the neural circuitry involved in maternal care and social behavior has been well documented (77)(78)(79)(80)(81). In mice, both Oxtr -/dams and Oxtr -/virgin females take longer to retrieve pups and spend less time crouching over them in the pup retrieval test (68). In addition to aberrant maternal behavior, Oxtr -/males are more aggressive in the resident intruder test demonstrated by an increase in the number and duration of attacks (68). Our findings of increased threatening behaviors and decreased maternal care in Tet1 Δe4-/mice recapitulate the findings observed in Oxtr -/mice and support the dysregulation of Oxtr in causing these behavioral deficits. The reduced response to oxytocin receptor agonist (TGOT) stimulation in TET1-deficient hippocampal neurons provides further support for impaired function of OXTR in the brain of Tet1 Δe4-/mice. However, it may be noted that, in addition to downregulation of Oxtr, there are other dysregulated genes in our Tet1 Δe4-/mice that may confound the interpretation of behavioral phenotypes; therefore, further studies such as reexpression of OXTR in select brain regions of Tet1 Δe4-/mice may be warranted.
OXTR has been the focus of extensive genetic and epigenetic studies for its role in psychiatric diseases or morbid behaviors in humans. There are more than 20 studies focusing on analysis of 5mC in the different CpG sites within the CpG island in humans (Supplemental Figure 10) (14,17,(54)(55)(56)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92). The regions equivalent to the hypermethylated BS1 and BS2 in Tet1 mutant mice have been the focus in these studies. In most studies, only select CpG sites, ranging from 1-20, within the CpG island are analyzed (55,93). Significant associations have been reported between OXTR DNA hypermethylation in these CpG sites and a wide spectrum of morbid behaviors and neuropsychiatric disease, such as ASD, schizophrenia, bipolar, OCD, and postpartum depression. Specifically, hypermethylation of a specific CpG site -934 of OXTR is reported in human postmortem autism brain tissues and is associated with schizophrenia, social response, and perception to anger and fear in human brains (14,55,56,93). The hypermethylation is typically associated with the reduced expressions of OXTR in the majority of the studies, but the opposite directions are also observed in a few CG sites. Until now, any mechanism underlying the change of methylation of OXTR in these studies was entirely unknown. Our finding provides the first evidence to our knowledge of a potential mechanism for OXTR hypermethylation and suggests that TET1 implicates the underlying mechanism of hypermethylation of human OXTR found in neuropsychiatric diseases.
The overall transcriptional regulation and isoform structure between human and mice are predicted to be highly conserved (5,6,67). The Oxtr transcript structure suggests a necessity of performing a reanalysis of results from existing epigenetic studies of OXTR in human, as the differences in methylation at unique CpG sites are likely to have different functional consequence on isoform-specific OXTR expression. In addition, multiple human studies have found strong associations between genomic variants in OXTR and ASD (13,(94)(95)(96). Our findings provide valuable information to reevaluate whether different OXTR isoforms are affected in these studies. Of particular interest, our finding that Oxtr is hypermethylated in tissues from all germ layers supports the use of peripheral blood in neurodevelopmental studies. Furthermore, our finding that TET1 has a dosage-sensitive effect on Oxtr methylation is particularly interesting, considering that heterozygous TET1 mutation has been reported in individuals with ASD (97). Altogether, our discovery of the involvement of TET1 in the epigenetic regulation of Oxtr and the complex transcriptional structure of Oxtr provide insights into understanding the role of OXTR in brain function and in neuropsychiatric disorders. The region-specific hypermethylation in the CpG island and isoform-specific reduction of Oxtr in Tet1 mutant mice will guide the experimental design for future OXTR epigenetic and genetic studies in human neuropsychiatric disorders.

Methods
Detailed experimental procedures can be found in Supplemental Methods.
Generation of Tet1 Δe4-/mice. For gene targeting and Southern analysis, we created a floxed construct of Tet1 exon 4 using the recombineering method described by Liu and colleagues (98). The 129SvEv BAC clone (bMQ-258L12) covering the Tet1 gene was first identified in silico using the Ensembl mouse genome browser (www.ensembl.org) and obtained from Geneservice (www.geneseservice.uk.com) (99). A 13.7-kb genomic fragment containing exon 4 of the Tet1 gene was retrieved into a plasmid from the BAC clone. A neomycin (neo) selectable marker flanked by flippase recognition target (FRT) sites was inserted into the plasmid. Two loxP sites were inserted into the plasmid flanking exon 4. The 5′ loxP was introduced at the genomic site of 62,304,376 bp, and the 3′ loxP/neo cassette at 62,300,243 bp (www.genome.ucsc.edu; mm9). The finalized plasmid was linearized with NotI and electroporated into R1 129/Sv mouse ESCs at the Duke Neurotransgenic Laboratory. Neo-resistant colonies were picked after 7-8 days of selection, and correctly recombinant clones were identified by Southern blot analysis using both a 5′ and 3′ probe. Tet1 e4+/f ESCs were injected into blastocysts to produce the chimeric male mice. These were bred with CMV-Cre females (The Jackson Laboratory, stock no. 006054) to obtain germline transmission of the Tet1-deleted allele. Tet1 Δe4+/mice were backcrossed to C57BL/6J mice (The Jackson Laboratory, stock no. 000664) for more than 6 generations (N6) before molecular experiments and behavioral analysis. We segregated out a naturally occurring mutation in the Disc1 gene in the 129R1 mouse strain from which ESCs were derived during the backcrossing. Multiple cohorts of Tet1 Δe4-/mice and WT control (Tet1 +/+ ) littermates were obtained from heterozygous breeding. All mice used for molecular experiments were between 5-10 weeks of age and were between 2-6 months for behavioral tests. Mice were sex-matched for all experiments.
Bisulfite sequencing. DNA (2 μg) from each sample was bisulfite-converted using the EpiTect bisulfite kit (Qiagen) according to the manufacturer's protocol. Bisulfite-converted DNA was amplified in 40 cycles using EpiMark Hot Start Taq DNA polymerase (New England Biolabs). Specific primers and annealing temperatures are listed in Supplemental Methods. PCR fragments were recovered by gel excision and cloned into the pGEM-T easy vector (Promega). Typically, 16 clones from each PCR product were sequenced with M13R primers at Beckman Coulter Genomics or EtonBio. A minimum of 10-12 clones with high-quality sequencing was obtained for each sample. Only the sequence products with bisulfite C-T conversion rate >99% were included for the data analysis, and we were able to achieve this conversion rate in most of experiments performed. Sequencing reads were analyzed using the online platform BISMA using default analysis parameters: Lower threshold conversion rate 99%, lower threshold sequence identity 90%, upper threshold of N-sites at cytosine position 20%, upper threshold gaps allowed 20% (100).
Field potential recording. Six-to 8 week-old mice were used for the LTP experiment. The hippocampus was cut in transverse sections at 400 μm in the slicing solution containing 75 mM sucrose, 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 10 mM glucose, 7 mM MgCl 2 , and 0.5 mM CaCl 2 . Slices were recovered at least 2 hours at 30°C in ACSF containing 124 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 10 mM glucose, 1 mM MgCl 2 , and 2 mM CaCl 2 . In the submersion recording chamber (30°C), a glass recording electrode (1-3 MΩ) filled with artificial cerebrospinal fluid (ACSF) was placed in CA1 radiatum. At baseline, stimulations (200 μsec, DS301 or Isoflex) were applied to the Schaffer collaterals every 30 seconds with a concentric bipolar tungsten electrode. The input-output relationship was obtained from increasing stimulation intensities (intervals of 5, 10, 15, 20, 25, 30, 40, 50, 60, 100 μA). Paired-pulse ratios were obtained from the field excitatory postsynaptic potential (fEPSP) slope of the second to the first stimulus at given interstimulus intervals (25-2,000 ms). For LTP, the stimulus strength was adjusted to evoke a fEPSP at half of the maximal response. After recording a stable baseline for 20 minutes (less than 5 % drift), high-frequency stimulation (100 Hz, 1 second) was applied. For the next 60 minutes, activity to a single pulse was recorded. The slope at 55-60 minutes was compared with the preconditioning baseline response (last 5 minutes of baseline). Values are expressed as means ± SEM.
Accession numbers. Oxtr mRNA isoform sequencing data has been deposited in GenBank under accession numbers KU686795-KU686801. RNA-seq and DNA methylome data have been deposited in GEO under accession number GSE99674.
Statistics. The data were analyzed with SPSS 21 (SPSS Inc.) or GraphPad Prism and expressed as mean ± SEM. Simple comparisons between Tet1 Δe4-/and Tet1 +/+ mice without regard to sex were conducted with independent t tests (Student's t test, 2-tailed). For behavioral data, when comparisons between genotypes were made for within-subject measurements across different phases of the same test (e.g., test days, locations within a test arena, or different intensities of stimuli), the data were analyzed with repeated measures 2-way ANOVA (RMANOVA). For 2-way RMANOVAs, a Tukey's correction for multiple comparisons was applied for post-hoc analysis. Statistical significance was defined as P < 0.05. Sample sizes were based on previous experience with similarly designed experiments.
Study approval. All experiments were conducted with protocols approved by the IACUC at Duke University. The animal studies presented in this paper have been approved by Duke Animal Care Committee with protocol number of A137-17-06.

Author contributions
AJT, XL, XK, and YJ conceived the experiments. AJT, XL, and XW created the Tet1 mice and ESCs. AJT, MWT, XC, XW, SX, and YK performed the molecular experiments. PW analyzed the RNA-seq data. WZ and WX performed the methylome experiment. AJT, ALB, LJD, and SKS performed behavioral experiments and ALB performed the statistical analyses. LC performed the slice physiology. AJT, MWT, SG, and YJ wrote the manuscript.


extfieldsofstudy: Chemistry Medicine

text:
Inhibitory Effects of Antimicrobial Photodynamic Therapy with Curcumin on Biofilm-Associated Gene Expression Profile of Aggregatibacter actinomycetemcomitans.

Objectives: Periodontitis is an inflammation of periodontal tissues that is caused by the biofilm of periodontal pathogens. Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is an opportunistic periodontopathogen that can be the cause of periodontal diseases via fimbriae as a virulence factor. In this study, we aimed to determine the expression level of A. actinomycetemcomitans rcpA gene as a virulence factor associated with biofilm formation after antimicrobial photodynamic therapy (aPDT) as a relatively new therapeutic modality. Materials and Methods: To determine sub-lethal doses of aPDT against A. actinomycetemcomitans ATCC 33384 strain, we used curcumin (CUR) as a photosensitizer at a final concentration of 40 μmol/ml, which was excited with a light-emitting diode (LED) at the wavelength of 450 nm. Quantitative real-time polymerase chain reaction (qRT-PCR) was then applied to monitor rcpA gene expression in A. actinomycetemcomitans. Results: 10–40 μmol/ml of CUR caused a significant reduction in the growth of A. actinomycetemcomitans compared to control group (P<0.05). Also, the cell viability of A. actinomycetemcomitans was significantly decreased after more than four minutes of LED irradiation. Therefore, the sub-lethal dose of aPDT against A. actinomycetemcomitans was 5 μmol/ml of CUR with three minutes of LED irradiation at a fluency of 180–240 J/cm2, which reduced the expression of the rcpA gene by approximately 8.5-fold. Conclusions: aPDT with CUR leads to decreased cell survival and virulence of A. actinomycetemcomitans. Thus, CUR-aPDT can be used as an alternative approach for the successful treatment of periodontitis in vivo.

INTRODUCTION
Periodontitis is an infection-induced inflammatory disease caused by multispecies biofilm models of periodontal pathogens [1]. Biofilms are surfaceattached microbial complex communities that are embedded in a matrix of extracellular polymeric substances [2]. Microbial biofilms are resistant to antimicrobial agents and can enhance the survival of the microbiota [3]. Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), an opportunistic periodontopathogen with various virulence factors, is able to resist the clearance attempts because of its protective extracellular matrix and due to the presence of resistant cells [4]. The tight-adherence (tad) gene locus in A. actinomycetemcomitans is necessary for surface attachment and biofilm formation. Different proteins encoded by the tad locus form A. actinomycetemcomitans fimbriae. Rough colony protein A (RcpA), a multimeric complex in th www.jdt.tums.ac.ir outer membrane of A. actinomycetemcomitans, forms a secreting channel that secretes the fimbriae components to outside the outer membrane [5]. The rcpA is a virulence factor that is known to be particularly important in A. actinomycetemcomitans isolates to form tight biofilms. It allows for interactions with host epithelial cells, which can lead to an up-regulation of biofilm-associated bacterial genes [6]. Several studies have suggested that antimicrobial photodynamic therapy (aPDT), also known as photochemotherapy and photoactivated disinfection [7], is a safe alternative strategy for periodontal therapy and can significantly change the cell survival of A. actinomycetemcomitans [8]. During aPDT, a photosensitizing chemical substance and a specific wavelength of light, used in conjunction with molecular oxygen, elicit cell death in microorganisms [7]. Recently, curcumin (CUR), a new class of photosensitizers, has been introduced for aPDT. CUR [1E,6E-1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione] is a yellow dye (also known as the spice turmeric) isolated from Curcuma longa. Recent investigations have shown that CUR is a potent photoactivatable substance that exhibits a variety of pharmacological properties such as antimicrobial, anti-inflammatory, anticancer, and antitumor activities [9,10]. To date, no data are available on the effects of aPDT with CUR, as a photosensitizer, on the expression of A. actinomycetemcomitans virulence factors. In the current study, we report the results of the effects of CUR-mediated aPDT on the expression of rcpA gene in A. actinomycetemcomitans cells surviving the PDT.

Determination of sub-lethal doses of CUR, light source, and aPDT:
The sub-minimum inhibitory concentration (sMIC) of CUR against A. actinomycetemcomitans, which is defined as a sub-lethal dose, was determined based on a previous study [11]. Briefly, 100 µl of 2X BHI broth was added to the wells of a roundbottom 96-well microplate (TPP AG, Trasadingen, Switzerland), and 100 µl of CUR was poured into the wells of the first column and was diluted two-fold stepwise. Afterward, 100 µl of A. actinomycetemcomitans suspension with 1.5×10 6 colony-forming units (CFUs)/ml was added to each well. In this study, the wells containing only A. actinomycetemcomitans suspension and the wells containing BHI broth without A. actinomycetemcomitans suspension or CUR were used as positive and negative controls, respectively. The microplate was incubated in the dark for 5 minutes at room temperature (25±2°C) in microaerophilic conditions. Next, 10 μl from the contents of each well was cultured in an enriched BHI agar plate which its components were described above. The plates were incubated for 48 hours at 37°C www.jdt.tums.ac.ir May 2018; Vol.15, No.3

Fig. 3:
The melting curve profiles generated by real-time amplification to assess potential primer-dimer artifacts (or non-specific PCR product). a.16S rRNA, b.rcpA in microaerophilic conditions. FUs/ml were determined based on the Miles and Misra method [12]. The sub-lethal dose of LED irradiation time against A. actinomycetemcomitans was determined according to a previous study [11]. In this method, 300 µl of the free-floating A. actinomycetemcomitans in the planktonic suspension at the final concentration of 1.5×10 5 CFU/ml was placed inside the microplate's wells. The LED in continuous mode was applied with an output power of 1000-1400 mW/cm 2 for 1, 2, 3, 4, and 5 minutes with energy densities of 60-80, 120-168, 180-240, 252-336, and 300-420 J/cm 2 , respectively. The LED probe was fixed at a 1-mm distance above each well's surface. A black paper was placed under the microplate to prevent beam reflection from the tabletop during LED irradiation. Calculations of A. actinomycetemcomitans CFUs/ml were performed according to the method mentioned above. Eventually, sub-lethal doses of aPDT were specified based on a previous study [13]. In brief, 100 µl of 2X BHI broth was added to the wells of the round-bottom 96-well microplate, and 100 µl of 2X MIC CUR was then added and serially diluted two-fold to 1.8 MIC. Finally, 100 µl of A. actinomycetemcomitans suspension with the concentration of 1.5×10 6 CFU/ml was poured into each well. The microplate was incubated for 5 minutes in the dark at room temperature in microaerophilic conditions before LED irradiation. The treated A. actinomycetemcomitans suspension in the well was exposed to the sub-lethal dose of LED irradiation time. Sub-lethal doses of aPDT were determined according to the methods mentioned previously [13]. The flowchart of the experimental steps is shown in Figure 1.

Sub-lethal doses of aPDT against A. actinomycetemcomitans isolates:
After determining the sub-lethal doses of aPDT in the previous section, the A. actinomycetemcomitans isolate was exposed to the sub-lethal dose of CUR and to the sub-lethal dose of LED irradiation time. The untreated A. actinomycetemcomitans isolate which did not receive sub-lethal doses of aPDT was used as a control group.

RNA preparation and complementary DNA (cDNA) synthesis:
The total RNA of treated and untreated A. actinomycetemcomitans isolates was extracted by using an RNA Extraction Kit (GeneAll Hybrid-RTM RNA purification; GeneAll Biotechnology Co., Ltd., Korea) in accordance with the manufacturer's recommendations. NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 260 nm and 280 nm (A260/A280 ratio) was used to evaluate the quality of the total extracted RNA. The total extracted RNA was treated by RNasefree DNase I (Thermo Fisher Scientific GmbH, Bremen, Germany) to eliminate the genomic DNA from RNA that could remain after the extraction. First-strand cDNA was then synthesized through random hexamer primed reactions by using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol.

Quantitative real-time polymerase chain reaction (qRT-PCR) and gene expression analysis:
The primers for A. actinomycetemcomitans were determined based on rcpA and comparative 16S ribosomal RNA (16S rRNA) gene sequences by utilizing Primer3 Input version 0.4.0 online software (http://frodo.wi.mit.edu/primer3/). Each nucleotide sequence was further evaluated by using the nucleotide algorithm of the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/). Also, the primer specificity was determined by melting curve analysis. The qRT-PCR assays were performed in triplicate by the Line-GeneK Real-Time PCR Detection System (Bioer Technology Co., Ltd., Hangzhou, China). qRT-PCR process contained the following reaction components in a 20-μl reaction: 1 μl of cDNA (corresponding to the cDNA transcribed from approximately 10 ng of RNA), 10 μl of SYBR Premix Ex Taq II (2x) (Takara Bio Inc., Otsu, Japan), 1 μl of forward and 1μl of reverse primer, which are shown in Table 1, and 7 μl of sterile distilled water. The thermal cycling conditions for qRT-PCR (25-μl reaction volume) included an initial denaturation of 5 minutes at 95°C, followed by 35 cycles at 95°C for 10 seconds, at 59°C for 15 seconds, and at 72°C for 15 seconds.

Statistical analysis:
One-way analysis of variance (ANOVA) and Tukey's post-hoc tests were performed to statistically analyze the data. The changes in the expression level of target gene were analyzed by using the method adopted by Livak and Schmittgen [14]. All the experiments were done in triplicate, and P-values lower than 0.05 were considered statistically significant.

RESULTS
According to the results of our study, CUR at 10-40 µmol/ml concentrations significantly reduced the cell survival of A. actinomycetemcomitans isolates from 50.5% to 79.1% compared to control group (untreated bacteria; P<0.05, Fig.  2a), whereas there was no statistically significant reduction when the concentration of CUR was increased from 0.3 to 5 µmol/ml. Therefore, the MIC and maximal sub-MIC doses of CUR against A. actinomycetemcomitans were found to be 5 and 2.5 µmol/ml, respectively. According to Figure 2b, our assays revealed that the cell viability of A. actinomycetemcomitans isolates was significantly reduced (30.8%) after 5 minutes of LED exposure with 300-420 J/cm 2 energy density. There was no decrease in the count of A. actinomycetemcomitans cells in the www other LED radiation times (P>0.05). Therefore, the sub-lethal dose of LED irradiation time was four minutes with an output light energy of 252-336 J/cm 2 . According to the results presented in Figure 2c, the maximal CUR-mediated sub-lethal dose of aPDT which exhibited a non-significant reduction in A. actinomycetemcomitans cell count was 1.2 µmol/ml of CUR plus three minutes of irradiation time at a fluency of 180-240 J/cm 2 . The results of each primer's melting curve in Figure 3 show that the presence of a single curve for each primer implies the formation of a single product, which shows the specificity of the primers for target genes. As shown in Figure 4, maximal sub-lethal doses of aPDT presented a significant 8.5-fold reduction of rcpA in A. actinomycetemcomitans cells and can be effective in reducing the biofilm formation of A. actinomycetemcomitans isolates. Overall, rcpA appears to play a critical role in the colonization of A. actinomycetemcomitans and in the formation of biofilms [5]. Interestingly, the microbiota in the biofilm phase are up to 1000-fold more tolerant and/or resistant to antimicrobial agents compared to the planktonic phase [19]. Also, the transformation efficiency rates can be 10-to 600-fold higher in biofilms than in planktonic cells [20].

DISCUSSION
A. actinomycetemcomitans can create persistent infections in periodontal regions and can promote resistance to antimicrobial agents and periodontal treatment via biofilm formation [19]. Therefore, the development of a new antimicrobial approach with fewer complications is necessary when there is a possibility of acquired resistance to antimicrobial elements by A. actinomycetemcomitans.
Previous studies have shown that aPDT, as a noninvasive therapeutic modality, is a new promising strategy to eliminate pathogenic microbiota [21][22][23]. During aPDT, a pharmacologically inert chromophore called a photosensitizer is used with a low-power laser of an appropriate wavelength. The photosensitizer is stimulated through exposure to light. In the presence of oxygen, the excited photosensitizer can release reactive nitrogen species (RNS) and oxygen species (ROS), such as superoxide, hydroxyl radicals, and hydrogen peroxide, which are harmful to cell membrane integrity and can cause biological death [21]. The antibacterial and antibiofilm effects of aPDT with different photosensitizers have been previously assessed. Haag et al [24] evaluated the One of the limitations of this study was in-vitro analysis without in-vivo assays. Further studies should be conducted on CUR-aPDT. Also, invivo evaluations are needed to confirm the clinical relevance of these results. In addition, CUR needs to be further improved for clinical use.

CONCLUSION
The results of the present study indicate that CURmediated aPDT shows antibacterial potential against A. actinomycetemcomitans and can repress the expression level of rcpA which is a gene involved in biofilm formation. It can be concluded that CUR-aPDT is a useful alternative strategy for periodontal therapy.