Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function.

Neuron-microglia interactions dictate the development of neuronal circuits in the brain. However, the factors that support and broadly regulate these processes across developmental stages are largely unknown. Here, we find that IL34, a neuron-derived cytokine, is upregulated in development and plays a critical role in supporting and maintaining neuroprotective, mature microglia in the anterior cingulate cortex (ACC) of mice. We show that IL34 mRNA and protein is upregulated in neurons in the second week of postnatal life and that this increase coincides with increases in microglia number and expression of mature, homeostatic markers, e.g., TMEM119. We also found that IL34 mRNA is higher in more active neurons, and higher in excitatory (compared to inhibitory) neurons. Genetic KO of IL34 prevents the functional maturation of microglia and results in an anxiolytic phenotype in these mice by adulthood. Acute, low dose blocking of IL34 at postnatal day (P)15 in mice decreased microglial TMEM119 expression and increased aberrant microglial phagocytosis of thalamocortical synapses within the ACC. In contrast, viral overexpression of IL34 early in life (P1-P8) caused early maturation of microglia and prevented microglial phagocytosis of thalamocortical synapses during the appropriate neurodevelopmental refinement window. Taken together, these findings establish IL34 as a key regulator of neuron-microglia crosstalk in postnatal brain development, controlling both microglial maturation and synapse engulfment.

Zika purified inactivated virus (ZPIV) vaccine reduced vertical transmission in pregnant immunocompetent mice.

Zika virus (ZIKV) is a significant threat to pregnant women and their fetuses as it can cause severe birth defects and congenital neurodevelopmental disorders, referred to as congenital Zika syndrome (CZS). Thus, a safe and effective ZIKV vaccine for pregnant women to prevent in utero ZIKV infection is of utmost importance. Murine models of ZIKV infection are limited by the fact that immunocompetent mice are resistant to ZIKV infection. As such, interferon-deficient mice have been used in some preclinical studies to test the efficacy of ZIKV vaccine candidates against lethal virus challenge. However, interferon-deficient mouse models have limitations in assessing the immunogenicity of vaccines, necessitating the use of immunocompetent mouse pregnancy models. Using the human stat2 knock-in (hSTAT2KI) mouse pregnancy model, we show that vaccination with a purified formalin-inactivated Zika virus (ZPIV) vaccine prior to pregnancy successfully prevented vertical transmission. In addition, maternal immunity protected offspring against postnatal challenge for up to 28 days. Furthermore, passive transfer of human IgG purified from hyper-immune sera of ZPIV vaccinees prevented maternal and fetal ZIKV infection, providing strong evidence that the neutralizing antibody response may serve as a meaningful correlate of protection.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Microbial modulation via cross-fostering prevents the effects of pervasive environmental stressors on microglia and social behavior, but not the dopamine system.

Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglial morphology and gene expression as well as decreased dopamine receptor expression and dopaminergic fiber input in the nucleus accumbens (NAc). Importantly, the gut-brain axis has been implicated in ASD, and both microglia and the dopamine system are sensitive to the composition of the gut microbiome. In line with this, we find that the composition of the gut microbiome and the structure of the intestinal epithelium are significantly shifted in DEP/MS-exposed males. Excitingly, both the DEP/MS-induced social deficits and microglial alterations in males are prevented by shifting the gut microbiome at birth via a cross-fostering procedure. However, while social deficits in DEP/MS males can be reversed by chemogenetic activation of dopamine neurons in the ventral tegmental area, modulation of the gut microbiome does not impact dopamine endpoints. These findings demonstrate male-specific changes in the gut-brain axis following DEP/MS and suggest that the gut microbiome is an important modulator of both social behavior and microglia.

Prenatal opioid exposure inhibits microglial sculpting of the dopamine system selectively in adolescent male offspring.

The current opioid epidemic has dramatically increased the number of children who are prenatally exposed to opioids, including oxycodone. A number of social and cognitive abnormalities have been documented in these children as they reach young adulthood. However, little is known about the mechanisms underlying developmental effects of prenatal opioid exposure. Microglia, the resident immune cells of the brain, respond to acute opioid exposure in adulthood. Moreover, microglia are known to sculpt neural circuits during typical development. Indeed, we recently found that microglial phagocytosis of dopamine D1 receptors (D1R) in the nucleus accumbens (NAc) is required for the natural developmental decline in NAc-D1R that occurs between adolescence and adulthood in rats. This microglial pruning occurs only in males, and is required for the normal developmental trajectory of social play behavior. However, virtually nothing is known as to whether this developmental program is altered by prenatal exposure to opioids. Here, we show in rats that maternal oxycodone self-administration during pregnancy leads to reduced adolescent microglial phagocytosis of D1R and subsequently higher D1R density within the NAc in adult male, but not female, offspring. Finally, we show prenatal and adult behavioral deficits in opioid-exposed offspring, including impaired extinction of oxycodone-conditioned place preference in males. This work demonstrates for the first time that microglia play a key role in translating prenatal opioid exposure to changes in neural systems and behavior.

Efficacy of an inactivated Zika vaccine against virus infection during pregnancy in mice and marmosets.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Tissue Flow Induces Cell Shape Changes During Organogenesis.

In embryonic development, cell shape changes are essential for building functional organs, but in many cases, the mechanisms that precisely regulate these changes remain unknown. We propose that fluid-like drag forces generated by the motion of an organ through surrounding tissue could generate changes to its structure that are important for its function. To test this hypothesis, we study the zebrafish left-right organizer, Kupffer's vesicle (KV), using experiments and mathematical modeling. During development, monociliated cells that comprise KV undergo region-specific shape changes along the anterior-posterior axis that are critical for KV function: anterior cells become long and thin, whereas posterior cells become short and squat. Here, we develop a mathematical vertex-like model for cell shapes that incorporates both tissue rheology and cell motility and constrain the model parameters using previously published rheological data for the zebrafish tailbud as well as our own measurements of the KV speed. We find that drag forces due to dynamics of cells surrounding KV could be sufficient or work in concert with previously identified mechanisms to drive KV cell shape changes during KV development. More broadly, these results suggest that cell shape changes during embryonic development and beyond could be driven by dynamic forces not typically considered in models or experiments.

Vertebrate myosin 1d regulates left-right organizer morphogenesis and laterality.

Establishing left-right asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia-driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event. Here, we report that myosin 1d (myo1d) is essential for establishing left-right asymmetry in zebrafish. Using super-resolution microscopy, we show that the zebrafish LRO, Kupffer's vesicle (KV), fails to form a spherical lumen and establish proper unidirectional flow in the absence of myo1d. This process requires directed vacuolar trafficking in KV epithelial cells. Interestingly, the vacuole transporting function of zebrafish Myo1d can be substituted by myosin1C derived from an ancient eukaryote, Acanthamoeba castellanii, where it regulates the transport of contractile vacuoles. Our findings reveal an evolutionary conserved role for an unconventional myosin in vacuole trafficking, lumen formation, and determining laterality.

Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer's vesicle.

How epithelial cell behaviors are coordinately regulated to sculpt tissue architecture is a fundamental question in biology. Kupffer's vesicle (KV), a transient organ with a fluid-filled lumen, provides a simple system to investigate the interplay between intrinsic cellular mechanisms and external forces during epithelial morphogenesis. Using 3-dimensional (3D) analyses of single cells we identify asymmetric cell volume changes along the anteroposterior axis of KV that coincide with asymmetric cell shape changes. Blocking ion flux prevents these cell volume changes and cell shape changes. Vertex simulations suggest cell shape changes do not depend on lumen expansion. Consistent with this prediction, asymmetric changes in KV cell volume and shape occur normally when KV lumen growth fails due to leaky cell adhesions. These results indicate ion flux mediates cell volume changes that contribute to asymmetric cell shape changes in KV, and that these changes in epithelial morphology are separable from lumen-generated forces.