An investigation into the potential neurobiological constructs and long-term biobehavioural effects of perinatal combined antiretroviral therapy (cART)-exposure in the deer mouse

MSc (Pharmacology), North-West University, Potchefstroom CampusSouth Africa has one of the largest combination antiretroviral treatment (cART1) programs in the world with an estimated 7.52 million individuals that were living with human immunodeficiency virus (HIV2) by the end of 2018. One meaningful development of this program, i.e. a treat-all approach, ensures that even pregnant women, regardless of CD43 count, have access to cART. This led to a reduction in the mother-to-child—or vertical—HIV transmission rate to below 2 %. Despite the fact that cART successfully reduces the HIV viral load, it is known to have HIV-unrelated adverse physiological effects, i.e. oxidative stress and proinflammatory responses. In turn, oxidative stress and central nervous system (CNS4) pro-inflammatory responses, either alone or presenting concurrently, have been associated with the manifestation, altered prognosis and poor treatment response of psychiatric conditions, e.g. major depressive disorder (MDD5) and bipolar disorder (BD6). Against this background, infants born HIV-uninfected but exposed to both HIV and cART in-utero (iHEU)7 and perinatally, i.e. the weeks immediately before and after birth, may be especially at risk. Observational studies indicate that some of these children progress to present with increased morbidities, including neuropsychological difficulties. Indeed, considering the neurodevelopmental window, during which neurulation, cell proliferation, axonal outgrowth and synaptogenesis occur, it is likely that infants of mothers that were treated with cART throughout pregnancy and the breast-feeding period, may be at risk for developing biobehavioral complications during the later stages of life. However, the extent to which perinatal cART exposure may result in long-term biobehavioral aberrations remains to be established, since the long-term study of children born from HIV-positive mothers, remains problematic. Therefore, the broad aim of this work was to investigate potential cART (i.e. a combination of tenofovir and emtricitabine)-induced biobehavioral alterations, as it pertains to oxidative stress (as measured by reduced to oxidized glutathione ratio; GSH/GSSG8), immune signaling (as reflected by both pro-inflammatory and anti-inflammatory cytokines), and behavioral expression over the different developmental phases of life in an uninfected and validated naturalistic animal model of a psychiatric phenotype, i.e. compulsive-like behavior as expressed by deer mice (Peromyscus maniculatus bairdii). This model system was chosen for this purpose, since a subpopulation, i.e. 30 – 35 %, of deer mice, irrespective of sex, naturalistically express persistent and repetitive large nest building (LNB1) behavior. Thus, by comparing the biobehavioral profile of normal nesting (NNB2) and LNB expressing animals that were perinatally exposed to either normal water or cART3, the immediate and long-term effects of said exposure in a standardized normal and compulsive-like cohort, can be explored. The GSH/GSSG4 ratio was chosen as a marker of oxidative stress, since perturbations in this system have been shown in clinical obsessive-compulsive disorder (OCD5) and in deer mice presenting with another compulsive-like phenotype, i.e. high motor stereotypy, before. It was thus of value to expand on these findings in the present investigation. In terms of inflammatory profile, we highlighted interleukin (IL6)-1β, IL-6, IL-10, IL-17, tumor necrosis factor-alpha (TNF-α7), and interferon-gamma (IFN-γ8) for investigation, as these have variably been implicated both in clinical OCD as well as in preclinical models of the disorder. Briefly, we hypothesized that perinatal cART exposure will induce oxidative stress and pro-inflammatory signaling in female and pregnant deer mice, as well as in their respective 3- and 14-week-old offspring. Given the reported associations between such aberrations and neuropsychiatric symptomology as alluded to above, we further expected that perinatal cART exposure would associate with an inflated and exacerbated degree of LNB expression later in life as compared to the behavior of the control-exposed cohort. * * * Three groups of 20 deer mouse dams were exposed to either normal water (control) or cART (tenofovir 60 mg/kg/day and emtricitabine 40 mg/kg/day, n = 10 per exposure; ethical approval nr.: NWU9-00524-20-A5) as follows: Group 1, all non-pregnant, was exposed to either intervention for 21 days and euthanized prior to pairing. Groups 2 and 3, all pregnant, were exposed from 21 days prior to mating until the end of nursing. Pups born from Group 2 were euthanized at 21 days (3-week-old group), while Group 3 pups were raised until adulthood when compulsivity testing, i.e. nesting assessment, was performed (14-week-old group). After collecting the whole-brain tissues of all animals, samples were analyzed for the GSH/GSSG ratio and cytokine profile (IL-1β, IL-6, IL-10, IL-17, IFN-γ, and TNF-α). Our main findings were that 1) both pregnancy and cART1 significantly decreased the whole-brain GSH/GSSG2 ratios in dams, 2) cART-, compared to control-exposure, varyingly decreased the concentrations of most cytokines measured in non-pregnant and pregnant dams, 3) differences in cytokine expression between control- and cART-exposed offspring only became apparent by the age of 14 weeks, in that cART-exposed offspring presented with significantly lower IL3-10, but higher IL-17 and IFN-ƴ4 concentrations than their age-matched control-exposed counterparts, 4) that cART-, compared to control-exposed LNB-expressing animals, generated lower total nesting scores, and 5) that the whole-brain concentrations of most cytokines were higher in cART-exposed 14-week-old NNB5-, compared to LNB6-expressing offspring. * * * As it is difficult to distinguish between the effects of maternal cART administration and the effects of HIV itself on the long-term development of affected children in a human cohort, this investigation was designed as a longitudinal exploration of the effects of cART on oxidative stress, immune signaling and behavioral expression in a naturalistic model of compulsive-like behavioral expression. Given the main findings of this work alluded to above, we successfully showed that such interactions between indirect exposure to cART during the perinatal period and central nervous system oxidative and inflammatory processes, do indeed exist and that the deer mouse model is a useful framework in which to study said interactions. Specifically, we showed that perinatal cART exposure exacerbates oxidative stress and suppresses immune signaling in pregnant vs non-pregnant mice. We further showed that cART-exposed NNB- and LNB-expressing offspring differ in terms of their immunological response to said exposure, with the long-term pro-inflammatory effects of cART exposure being masked in LNB-expressing offspring. Collectively, this data highlights a dichotomous influence of cART on the immediate and long-term functioning of the immune system. We also showed that LNB uniquely associates with a perturbed brain-immune crosstalk fingerprint and that future investigations using the deer mouse model of compulsive-like behavior will be vital for an expanded understanding of related concepts.Master