Multidrug-resistant (MDR) bacterial pneumonias pose a critical threat to global public health. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is a leading cause of nosocomial-associated pneumonia, and an effective vaccine could protect vulnerable populations, including the elderly, immunocompromised, and those with chronic respiratory diseases. Highly heterogeneous outer membrane vesicles (OMVs), shed from Gram-negative bacteria, are studded with immunogenic lipids, proteins, and virulence factors. To overcome limitations in OMV stability and consistency, we described a believed to be novel vaccine platform that combines immunogenic OMVs with precision nanotechnology—creating a bacterial cellular nanoparticle vaccine candidate (CNP), termed Pa-STING-CNP, which incorporates an adjuvanted core that activates the STING (stimulator of interferon genes) pathway. In this design, OMVs are coated onto the surface of self-adjuvanted STING nanocores. Pa-STING CNP vaccination induced substantial antigen presenting cell recruitment and activation in draining lymph nodes, robust anti-Pseudomonas antibody responses, and provided protection against lethal challenge with the hypervirulent clinical P. aeruginosa isolate PA14. Antibody responses mediated this protection and provided passive immunity against the heterologous P. aeruginosa strain PA01. These findings provided evidence that nanotechnology can be used to create a highly efficacious vaccine platform against high priority MDR pathogens such as P. aeruginosa.

Background: Phosphoinositide 3-kinase is a potent target for cancer therapy due to its significant role in the regulation of cellular growth and proliferation. Dysregulation of the PI3k signaling cascade can constitutively activate growth pathways to trigger the progression of cancer, resulting in the development of multiple inhibitors as cancer therapeutics. Objectives: The wide array of cells expressing PI3k also include immune cells, and the inhibition of these receptors has shown promise in combating inflammation and infectious disease, a relationship we sought to examine further. Methods: We infected wild-type and PI3kγ knockout murine macrophages as well as PI3kγ inhibitor-treated THP-1 human macrophage-like cells with Staphylococcus aureus and quantified inflammation through gene expression analysis, protein secretion assays, and immunofluorescence imaging. Results: We observed that knockout of PI3kγ in murine macrophages alongside pharmacological inhibition through IPI549 treatment in THP-1 cells led to an NF-κB-driven suppression in transcription and release of inflammatory cytokines upon infection with methicillin-resistant Staphylococcus aureus. We were also able to confirm that this suppression of NF-κB translocation and subsequent decrease in inflammatory cytokine release did not compromise and even slightly boosted the bacterial killing ability. Conclusion: PI3k is primarily targeted for cancer therapies, but further exploration can also be carried out on its potential roles in treating bacterial infection.

Klebsiella pneumoniae (Kp) is a leading cause of hospital- and community-acquired infections and is increasingly resistant to last-line antibiotics, positioning it as a World Health Organization-designated priority pathogen. Vaccine development has been hindered by the extensive diversity of Kp exopolysaccharide capsules. We developed a cellular nanoparticle (CNP) platform in which bacterial outer membrane vesicles (OMVs) are stably coated onto a STING (stimulator of interferon genes)-adjuvanted nanoparticle core (CNP-KpSTING). The resulting ∼100-nm nanoparticles are homogenous and optimized for recruitment and activation of antigen-presenting cells in draining lymph nodes. CNP-KpSTING vaccination elicited robust antibody responses, enhanced neutrophil-mediated bacterial clearance, and conferred cross-protection against heterologous Kp capsule types in vitro and in lethal pneumonia models. Passive transfer of post-vaccination sera reduced bacterial burden and inflammatory cytokines in the lungs of infected mice. These findings establish CNP-KpSTING as a versatile platform that addresses key antigenic barriers inherent to Kp and support OMV-CNPs as a promising foundation for broadly protective vaccines against multidrug-resistant Gram-negative pathogens.

Purpose: This study investigated the differential gene expression of BMPs in chick retinal pigment epithelium (RPE) during recovery from short term exposure to optical defocus and form-deprivation (FD) treatments. Methods: 14-day old White-Leghorn chicks wore either monocular +10 or -10 D lenses, or diffusers for 2 or 48 h, after which eyes were allowed unobstructed vision for up to 96 h. Over this recovery period, refractive errors and choroidal thickness (ChT) were tracked using retinoscopy and high-frequency A-scan ultrasonography. Real-time PCR was used to examine the expression of BMP2, 4, and 7 genes in RPE samples collected 0, 15 min, 2, 24, 48, and 96 h after the termination of treatments. Expression levels in treated eyes and their contralateral control eyes were compared. Results: After the termination of the lens and diffuser treatments, eyes gradually recovered from induced shifts in refractive error. With all three treatments, ChT changes reached statistical significance after 48 h of treatment, be it thinning with the -10 D lens and diffuser treatments (-0.06 ± 0.03mm, p < 0.05; -0.11 ± 0.04 mm, p < 0.05, resp.), or thickening with the +10 D lens (0.31 ± 0.04 mm, p < 0.001). BMP2 gene expression was rapidly upregulated in eyes wearing the +10 D lens, being statistical significance after 2 h, as well as 48 h of treatment. With the 2 h treatment, the latter gene expression pattern persisted for 15 min into the recovery period, before decreasing to the same level as that of contralateral control eyes, with a short-lived rebound, i.e., upregulation, 24 h into the recovery period. With the longer, 48 h treatment, BMP2 gene expression decreased more gradually, from 739 ± 121% at the end of the treatment period, to 72 ± 14% after 48 h of recovery. Two and 48 h of both -10 D and FD treatments resulted in BMP2 gene expression downregulation, with the time taken for gene expression levels to fully recover varying with the duration of initial treatments. In both cases, BMP2 gene expression downregulation persisted for 15 min into the recovery period, but reversed to upregulation by 2 h. Similar gene expression patterns were also observed for BMP4, although the changes were smaller. Conclusions: The observed changes in BMP gene expression in chick RPE imply dynamic, albeit complex regulation, with the duration of exposure and recovery being critical variables for all three types of visual manipulations. This study provides further evidence for a role of the RPE as an important signal relay linking the retina to the choroid and sclera in eye growth regulation.

Staphylococcus aureus (SA) causes a broad range of invasive human diseases, increasingly complicated by antibiotic resistance. Macrophages (MΦ) play a central role in innate immunity, yet key aspects of their interaction with SA remain incompletely understood because of the pathogen’s diverse immune evasion strategies. Using multi-omics and systems-level analytics, we identified the E3 ubiquitin ligase cullin 4B (CUL4B) as a host factor that modulates MΦ responses to SA infection. CRISPR-mediated deletion of CUL4B in human THP-1 MΦ enhanced intracellular bacterial killing, reduced cell death, decreased caspase-1 activation, and lowered interleukin-1β production, consistent with altered inflammasome signaling. In a murine methicillin-resistant SA (MRSA) pneumonia model, CUL4B deficiency improved pulmonary bacterial clearance, reduced bacteremia, and decreased inflammatory tissue injury. Whole-lung and serum proteomics further revealed increased antimicrobial protein abundance and reduced markers of inflammatory damage. Together, these findings identify CUL4B as a regulator of MΦ inflammatory responses during MRSA infection and suggest a potential host-directed pathway that could be leveraged to improve outcomes against this major pathogen.