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BackgroundGlobally, over 16 million children were exposed to HIV during pregnancy but remain HIV-free at birth and throughout childhood by 2022. Children born HIV-free (CBHF) have higher morbidity and mortality and poorer neurodevelopment in early life compared to children who are HIV-unexposed (CHU), but long-term outcomes remain uncertain. We characterised school-age growth, cognitive and physical function in CBHF and CHU previously enrolled in the Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial in rural Zimbabwe.Methods and findingsThe SHINE trial enrolled pregnant women between 2012 and 2015 across 2 rural Zimbabwean districts. Co-primary outcomes were height-for-age Z-score and haemoglobin at age 18 months (clinicaltrials.gov NCT01824940). Children were re-enrolled if they were aged 7 years, resident in Shurugwi district, and had known pregnancy HIV-exposure status. From 5,280 pregnant women originally enrolled, 376 CBHF and 2016 CHU reached the trial endpoint at 18 months in Shurugwi; of these, 264 CBHF and 990 CHU were evaluated at age 7 years using the School-Age Health, Activity, Resilience, Anthropometry and Neurocognitive (SAHARAN) toolbox. Cognitive function was evaluated using the Kaufman Assessment Battery for Children (KABC-II), with additional tools measuring executive function, literacy, numeracy, fine motor skills, and socioemotional function. Physical function was assessed using standing broad jump and handgrip for strength, and the shuttle-run test for cardiovascular fitness. Growth was assessed by anthropometry. Body composition was assessed by bioimpedance analysis and skinfold thicknesses. A caregiver questionnaire measured demographics, socioeconomic status, nurturing, child discipline, food, and water insecurity. We prespecified the primary comparisons and used generalised estimating equations with an exchangeable working correlation structure to account for clustering. Adjusted models used covariates from the trial (study arm, study nurse, exact child age, sex, calendar month measured, and ambient temperature). They also included covariates derived from directed acyclic graphs, with separate models adjusted for contemporary variables (socioeconomic status, household food insecurity, religion, social support, gender norms, caregiver depression, age, caregiver education, adversity score, and number of children's books) and early-life variables (length-for-age-Z-score) at 18 months, birthweight, maternal baseline depression, household diet, maternal schooling and haemoglobin, socioeconomic status, facility birth, and gender norms. We applied a Bonferroni correction for the 27 comparisons (0.05/27) with threshold of p < 0.00185 as significant. We found strong evidence that cognitive function was lower in CBHF compared to CHU across multiple domains. The KABC-II mental processing index was 45.2 (standard deviation (SD) 10.5) in CBHF and 48.3 (11.3) in CHU (mean difference 3.3 points [95% confidence interval (95% CI) 2.0, 4.5]; p < 0.001). The school achievement test score was 39.0 (SD 26.0) in CBHF and 45.7 (27.8) in CHU (mean difference 7.3 points [95% CI 3.6, 10.9]; p < 0.001); differences remained significant in adjusted analyses. Executive function was reduced but not significantly in adjusted analyses. We found no consistent evidence of differences in growth or physical function outcomes. The main limitation of our study was the restriction to one of two previous study districts, with possible survivor and selection bias.ConclusionsIn this study, we found that CBHF had reductions in cognitive function compared to CHU at 7 years of age across multiple domains. Further research is needed to define the biological and psychosocial mechanisms underlying these differences to inform future interventions that help CBHF thrive across the life-course.Trial registrationClinicalTrials.gov The SHINE follow-up study was registered with the Pan-African Clinical Trials Registry (PACTR202201828512110). The original SHINE trial was registered at NCT https://clinicaltrials.gov/study/NCT01824940.

Chemotherapy is a widely used treatment for a variety of solid and hematological malignancies. Despite its success in improving the survival rate of cancer patients, chemotherapy causes significant toxicity to multiple organs, including the skeleton, but the underlying mechanisms have yet to be elucidated. Using tumor-free mouse models, which are commonly used to assess direct off-target effects of anti-neoplastic therapies, we found that doxorubicin caused massive bone loss in wild-type mice, a phenotype associated with increased number of osteoclasts, leukopenia, elevated serum levels of danger-associated molecular patterns (DAMPs; e.g. cell-free DNA and ATP) and cytokines (e.g. IL-1β and IL-18). Accordingly, doxorubicin activated the absent in melanoma (AIM2) and NLR family pyrin domain containing 3 (NLRP3) inflammasomes in macrophages and neutrophils, causing inflammatory cell death pyroptosis and NETosis, which correlated with its leukopenic effects. Moreover, the effects of this chemotherapeutic agent on cytokine secretion, cell demise, and bone loss were attenuated to various extent in conditions of AIM2 and/or NLRP3 insufficiency. Thus, we found that inflammasomes are key players in bone loss caused by doxorubicin, a finding that may inspire the development of a tailored adjuvant therapy that preserves the quality of this tissue in patients treated with this class of drugs.

Osteoarthritis is the most common joint disease in the world with significant societal consequences but lacks effective disease-modifying interventions. The pathophysiology consists of a prominent inflammatory component that can be targeted to prevent cartilage degradation and structural defects. Intracellular metabolism has emerged as a culprit of the inflammatory response in chondrocytes, with both processes co-regulating each other. The role of glutamine metabolism in chondrocytes, especially in the context of inflammation, lacks a thorough understanding and is the focus of this work. We display that mouse chondrocytes utilize glutamine for energy production and anabolic processes. Furthermore, we show that glutamine deprivation itself causes metabolic reprogramming and decreases the inflammatory response of chondrocytes through inhibition of NF-κB activity. Finally, we display that glutamine deprivation promotes autophagy and that ammonia is an inhibitor of autophagy. Overall, we identify a relationship between glutamine metabolism and inflammatory signaling and display the need for increased study of chondrocyte metabolic systems.

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1β (IL-1β) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1β and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-κB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKγ/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions.

Abstract The NLRP3 inflammasome senses a variety of signals referred to as danger associated molecular patterns (DAMPs), including those triggered by crystalline particulates or degradation products of extracellular matrix. Since some DAMPs confer tissue-specific activation of the inflammasomes, we tested the hypothesis that bone matrix components function as DAMPs for the NLRP3 inflammasome and regulate osteoclast differentiation. Indeed, bone particles cause exuberant osteoclastogenesis in the presence of RANKL, a response that correlates with NLRP3 abundance and the state of inflammasome activation. To determine the relevance of these findings to bone homeostasis, we studied the impact of Nlrp3 deficiency on bone using pre-clinical mouse models of high bone turnover, including estrogen deficiency and sustained exposure to parathyroid hormone or RANKL. Despite comparable baseline indices of bone mass, bone loss caused by hormonal or RANKL perturbations is significantly reduced in Nlrp3 deficient than in wild type mice. Consistent with the notion that osteolysis releases DAMPs from bone matrix, pharmacologic inhibition of bone resorption by zoledronate attenuates inflammasome activation in mice. Thus, signals originating from bone matrix activate the NLRP3 inflammasome in the osteoclast lineage, and may represent a bone-restricted positive feedback mechanism that amplifies bone resorption in pathologic conditions of accelerated bone turnover.

Abstract Skeletal complications are common features of neonatal-onset multisystem inflammatory disease (NOMID), a disorder caused by NLRP3-activating mutations. NOMID mice in which NLRP3 is activated globally exhibit several characteristics of the human disease, including systemic inflammation and cartilage dysplasia, but the mechanisms of skeletal manifestations remain unknown. In this study, we find that activation of NLRP3 in myeloid cells, but not mesenchymal cells triggers chronic inflammation, which ultimately, causes growth plate and epiphyseal dysplasia in mice. These responses are IL-1 signaling-dependent, but independent of PARP1, which also functions downstream of NLRP3 and regulates skeletal homeostasis. Mechanistically, inflammation causes severe anemia and hypoxia in the bone environment, yet down-regulates the HIF-1α pathway in chondrocytes, thereby promoting the demise of these cells. Thus, activation of NLRP3 in hematopoietic cells initiates IL-1β-driven paracrine cascades, which promote abnormal growth plate development in NOMID mice.

The use of zebrafish larvae has aroused wide interest in the medical field for its potential role in the development of new therapies. The larvae grow extremely quickly and the embryos are nearly transparent which allows easy examination of its internal structures using fluorescent imaging techniques. Medical treatment of zebrafish larvae can directly influence its swimming behaviours. These behaviour changes are related to functional changes of central nervous system and transformations of the zebrafish body such as muscle mechanical power and force variation, which cannot be measured directly by pure experiment observation. To quantify the influence of drugs on zebrafish larvae swimming behaviours and energetics, we have developed a novel methodology to exploit intravital changes based on observed zebrafish locomotion. Specifically, by using an in-house MATLAB code to process the recorded live zebrafish swimming video, the kinematic locomotion equation of a 3D zebrafish larvae was obtained, and a customised Computational Fluid Dynamics tool was used to solve the fluid flow around the fish model which was geometrically the same as experimentally tested zebrafish. The developed methodology was firstly verified against experiment, and further applied to quantify the fish internal body force, torque and power consumption associated with a group of normal zebrafish larvae vs. those immersed in acetic acid and two neuroactive drugs. As indicated by our results, zebrafish larvae immersed in 0.01% acetic acid display approximately 30% higher hydrodynamic power and 10% higher cost of transport than control group. In addition, 500 μM diphenylhydantoin significantly decreases the locomotion activity for approximately 50% lower hydrodynamic power, whereas 100 mg/L yohimbine has not caused any significant influences on 5 dpf zebrafish larvae locomotion. The approach has potential to evaluate the influence of drugs on the aquatic animal’s behaviour changes and thus support the development of new analgesic and neuroactive drugs.

Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca) on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het)) and homozygotes (Cre+IKK2ca_f/f (KI)) showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers Indian hedgehog and alkaline phosphatase, and the early markers Aggrecan and type-II collagen were reduced in Cre+IKK2ca_w/f and Cre+IKK2ca_f/f mice. Altogether, the in-vitro, in vivo and ex-vivo evidence suggest that IKK2ca perturbs osteoblast and chondrocyte maturation and impairs skeletal development.

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1β. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1β and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue "spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.