Neonatal inflammation is common and has lasting consequences for adult health. We investigated the lasting effects of a single bout of neonatal inflammation on adult respiratory control in the form of respiratory motor plasticity induced by acute intermittent hypoxia, which likely compensates and stabilizes breathing during injury or disease and has significant therapeutic potential. Lipopolysaccharide-induced inflammation at postnatal day four induced lasting impairments in two distinct pathways to adult respiratory plasticity in male and female rats. Despite a lack of adult pro-inflammatory gene expression or alterations in glial morphology, one mechanistic pathway to plasticity was restored by acute, adult anti-inflammatory treatment, suggesting ongoing inflammatory signaling after neonatal inflammation. An alternative pathway to plasticity was not restored by anti-inflammatory treatment, but was evoked by exogenous adenosine receptor agonism, suggesting upstream impairment, likely astrocytic-dependent. Thus, the respiratory control network is vulnerable to early-life inflammation, limiting respiratory compensation to adult disease or injury.

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Neonatal inflammation is common and disrupts adult respiratory control, yet the mechanism(s) underlying the impairments are not well understood. Since microglia are key mediators of central inflammation and are important in the lasting effects of neonatal inflammation in non‐respiratory brain regions, we hypothesized lasting dysfunction in microglia underlie impairments in adult respiratory control. Specifically, we hypothesized neonatal inflammation would increase the number of and inflammatory gene expression in adult microglia. Since adult respiratory motor plasticity in the cervical spinal cord is impaired after neonatal inflammation, we analyzed immunomagnetically isolated microglia from the ventral cervical spinal cord. Interestingly, the number of microglia measured by flow cytometry calibrated with counting beads from adults after neonatal inflammation (LPS; 1 mg/kg, i.p.; postnatal day 4) were unchanged in males (LPS: 14,488 ± 4000, n=4; saline: 12,784 ± 4000, n=4, p=0.97) or females (LPS: 14,611 ± 4500, n=4; saline: 9,062 ± 4500, n=5, p=0.79). Additionally, the number of microglia was not significantly different between males and females (p>0.05). Despite no changes in microglia number, we hypothesized microglia function was impaired by neonatal inflammation. Preliminary transcriptomic analysis suggests neonatal inflammation in adult females (n=2) up‐regulated 7 genes and down‐regulated 1 gene, and in adult males (n=3) down‐regulated 1 gene. Gene ontology revealed these differentially expressed genes are associated with inflammatory signaling. While neonatal inflammation has no impact on the number of microglia, it sex‐dependently increases inflammatory‐related gene expression in females with surprisingly few changes in adult males. Thus, adult, female microglia likely contribute to the lasting impairments in respiratory control following neonatal inflammation. Further, the lasting impairments in adult males are likely mediated by a sex‐specific distinct mechanism. Understanding the sex‐specific inflammatory mechanisms impairing respiratory control is crucial to developing treatment strategies to promote breathing during ventilatory control disorders. Support or Funding Information Supported by NIH HL141249 (AGH)

Adults often encounter diverse inflammatory stimuli, yet only some adults are prone to breathing deficits after adult inflammation. The determinants of adult susceptibility to breathing deficits during inflammation is understudied and likely involves microglia, the resident immune cells of the central nervous system. In non‐respiratory control regions, neonatal inflammation primes adult microglia for exaggerated inflammatory responses to otherwise harmless inflammatory stimuli, such as adult subthreshold heterotypic inflammation (differing neonatal and adult inflammatory stimuli). Since male and female microglia have sexually dimorphic developmental trajectories, we hypothesized that neonatal inflammation would sex‐dependently prime adult microglia in respiratory control regions to subthreshold heterotypic inflammation, contributing to impaired adult breathing. Using flow cytometry to assess microglia number (%CD11bhighCD45low / homogenates) and priming, adult male medullary microglia after neonatal inflammation (LPS 1mg/kg, i.p.) and adult subthreshold heterotypic inflammation (polyIC 478ug/kg, i.p.) were primed (neonatal LPS + adult polyIC: 25±7% microglia n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9, p<0.05; neonatal saline + adult polyIC: 11±2% microglia, n=8, p<0.0001). In females, neonatal inflammation increased, but did not prime, medullary microglia to adult heterotypic inflammation (neonatal LPS + adult polyIC: 17±3% microglia, n=6; neonatal saline + adult saline: 11±1% microglia, n=6; neonatal saline + adult polyIC: 11±2% microglia, n=6, p<0.005; neonatal LPS + saline: 14±2% microglia, n=8, p>0.9). To test whether these primed microglia in respiratory control regions contribute to adult breathing deficits, breathing was assessed using plethysmography in adults after neonatal and adult heterotypic inflammation. Contrary to our hypothesis, neonatal and adult heterotypic inflammation did not impair adult eupneic breathing, the hypercapnic ventilatory response, or the hypoxic ventilatory response in adult males (p>0.9) or females (p>0.9). Therefore, adults after neonatal and adult heterotypic inflammation are able maintain breathing in response to low levels of inflammation despite primed medullary microglia. Primed microglia, however, are prone to augmented inflammatory responses and may induce significant deficits to breathing when faced with more severe inflammatory challenges.