Development of Pro-resolving and Pro-efferocytic Nanoparticles for Atherosclerosis Therapy.

Atherosclerosis is a major contributor to cardiovascular diseases with a high global prevalence. It is characterized by the formation of lipid-laden plaques in the arteries, which eventually lead to plaque rupture and thrombosis. While the current lipid-lowering therapies are generally effective in lowering the risk of cardiovascular events, they do not address the underlying causes of disease. Defective resolution of inflammation and impaired efferocytosis are the main driving forces of atherosclerosis. Macrophages recognize cells for clearance by the expression of "eat me" and "do not eat me" signals, including the CD47-SIRPα axis. However, the "do not eat me" signal CD47 is overexpressed in atherosclerotic plaques, leading to compromised efferocytosis and secondary necrosis. In this context, prophagocytic antibodies have been explored to stimulate the clearance of apoptotic cells, but they are nonspecific and impact healthy tissues. In macrophages, downstream of signal regulatory protein α, lie protein tyrosine phosphatases, SHP 1/2, which can serve as effective targets for selectively phagocytosing apoptotic cells. While increasing the efferocytosis targets the end stages of lesion development, the underlying issue of inflammation still persists. Simultaneously increasing efferocytosis and reducing inflammation can be effective therapeutic strategies for managing atherosclerosis. For instance, IL-10 is a key anti-inflammatory mediator that enhances efferocytosis via phosphoSTAT3 (pSTAT3) activation. In this study, we developed a combination nanotherapy by encapsulating an SHP-1 inhibitor (NSC 87877) and IL-10 in a single nanoparticle platform [(S + IL)-NPs] to enhance efferocytosis and inflammation resolution. Our studies suggest that (S + IL)-NPs successfully encapsulated both agents, entered the macrophages, and delivered the agents into intracellular compartments. Additionally, (S + IL)-NPs decreased inflammation by suppressing pro-inflammatory markers and enhancing anti-inflammatory mediators. They also exhibited the potential for improved phagocytic activity via pSTAT3 activation. Our nanomedicine-mediated upregulation of the anti-inflammatory and efferocytic responses in macrophages shows promise for the treatment of atherosclerosis.

The enduring effects of antimicrobials and lipopolysaccharide on the cellular mechanisms and behaviours associated with neurodegeneration in pubertal male and female CD1 mice.

Puberty is a sensitive developmental period during which stressors can cause lasting brain and behavioural deficits. While the acute effects of pubertal lipopolysaccharide (LPS) and antimicrobial (AMNS) treatments are known, their enduring impacts on neurodegeneration-related mechanisms and behaviours remain unclear. This study examined these effects in male and female mice. At five weeks old, mice received 200ul of either broad-spectrum antimicrobials or water through oral gavage twice daily for seven days. At six weeks of age, they received an intraperitoneal injection of either saline or LPS. Four weeks later, adult mice underwent neurodegeneration-related behavioural tests, including the rotarod, forepaw stride length, reversed grid hang, open field, and buried pellet tests. Two days after the final test, brain and ileal samples were collected. Results showed that female mice treated with both AMNS and LPS exhibited deficits in neuromuscular strength, while males treated with LPS alone showed increased anxiety-like behaviours. Males treated with AMNS alone had decreased sigma-1 receptor (S1R) expression in the cornu ammonis 1 (CA1) and dentate gyrus (DG), while females treated with both AMNS and LPS had decreased S1R expression. Additionally, males treated with either LPS or AMNS had lower glial-derived neurotrophic factor receptor alpha-1 (GFRA1) expression in the primary motor cortex (M1) than females. Mice treated with LPS alone had decreased GFRA1 expression in the DG and decreased S1R expression in the secondary motor cortex (M2). These findings suggest that pubertal AMNS and LPS treatments may lead to enduring changes in biomarkers and behaviours related to neurodegeneration.

CD46 expression in the central nervous system of male and female pubescent mice.

CD46 is a complementary regulatory protein ubiquitously expressed in human cells, controlling complement system activation. CD46 has further been identified to have several other functions including regulatory T cell induction and intestinal epithelial (IEC) barrier regulation. Activation of CD46 in the IEC can impact intestinal barrier permeability and immune system functioning. CD46 has only been identified in the spermatozoa and retina of mice. In other murine cells, the homologue CRRY is identified to function as the complementary regulator. Due to the identification of CRRY across other wild-type mouse cells and the development of mouse strains transgenic for human CD46, no recent research has been conducted to determine if CD46 is present in non-transgenic mouse strains. Therefore, the current study investigated if CD46 is expressed in the substantia nigra (SN) and caudate putamen (CP) of pubescent CD1 mice and examined the acute effects of pubertal antimicrobial and lipopolysaccharide (LPS) treatment on CD46 expression in the brain. As of 5 weeks of age, mice were administered mixed antimicrobial solution or water with oral gavage twice daily for 7 days. At 6 weeks of age, mice received an intraperitoneal injection of LPS or saline. Mice were euthanized 8 h post-injection and brain samples were collected. Our results indicate that pubescent CD-1 mice express CD46 in the SN and CP. However, LPS-treated mice displayed significantly less CD46 expression in the SN in comparison to saline-treated mice. Furthermore, males displayed more CD46 in the CP compared to females, regardless of LPS and antimicrobial treatments. Our data suggest CD46 is present in CD1 mice and that LPS and antimicrobial treatments impact CD46 protein expression in a sex-dependent manner. These results have important implications for the expression of CD46 in the mouse brain and the understanding of its role in immune system regulation.

The acute effects of antimicrobials and lipopolysaccharide on the cellular mechanisms associated with neurodegeneration in pubertal male and female CD1 mice.

Exposure to stressors during puberty can cause enduring effects on brain functioning and behaviours related to neurodegeneration. However, the mechanisms underlying these effects remain unclear. The gut microbiome is a complex and dynamic system that could serve as a possible mechanism through which early life stress may increase the predisposition to neurodegeneration. Therefore, the current study was designed to examine the acute effects of pubertal antimicrobial and lipopolysaccharide (LPS) treatments on the cellular mechanisms associated with neurodegenerative disorders in male and female mice. At five weeks of age, male and female CD-1 mice received 200 µL of broad-spectrum antimicrobials or water, through oral gavage, twice daily for seven days. Mice received an intraperitoneal (i.p.) injection of either saline or LPS at 6 weeks of age (i.e., pubertal period). Sickness behaviours were recorded and mice were euthanized 8 h post-injection. Following euthanasia, brains and blood samples were collected. The results indicated that puberal antimicrobial and LPS treatment induced sex-dependent changes in biomarkers related to sickness behaviour, peripheral inflammation, intestinal permeability, and neurodegeneration. The findings suggest that pubertal LPS and antimicrobial treatment may increase susceptibility to neurodegenerative diseases later in life, particularly in males.

The effects of antimicrobials and lipopolysaccharide on acute immune responsivity in pubertal male and female CD1 mice.

Exposure to stress during critical periods of development-such as puberty-is associated with long-term disruptions in brain function and neuro-immune responsivity. However, the mechanisms underlying the effect of stress on the pubertal neuro-immune response has yet to be elucidated. Therefore, the objective of the current study was to investigate the effect antimicrobial and lipopolysaccharide (LPS) treatments on acute immune responsivity in pubertal male and female mice. Moreover, the potential for probiotic supplementation to mitigate these effects was also examined. 240 male and female CD1 mice were treated with one week of antimicrobial treatment (mixed antimicrobials or water) and probiotic treatment (L. rhamnosis R0011 and L. helveticus R0052 or L. helveticus R0052 and B. longum R0175) or placebo at five weeks of age. At six weeks of age (pubertal stress-sensitive period), the mice received a single injection of LPS or saline. Sickness behaviours were assessed, and mice were euthanized 8 h post-injection. Brain, blood, and intestinal samples were collected. The results indicated that the antimicrobial treatment reduced sickness behaviours, and potentiated LPS-induced plasma cytokine concentrations and pro-inflammatory markers in the pre-frontal cortex (PFC) and hippocampus, in a sex-dependent manner. However, probiotics reduced LPS-induced plasma cytokine concentrations along with hippocampal and PFC pro-inflammatory markers in a sex-dependent manner. L. rhamnosis R0011 and L. helveticus R0052 treatment also mitigated antimicrobial-induced plasma cytokine concentrations and sickness behaviours. These findings suggest that the microbiome is an important modulator of the pro-inflammatory immune response during puberty.