Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy.

Here, we present a protocol to deliver nanoliter volumes of Toll-like receptor (TLR) agonist onto a culture of nuclear factor κB (NF-κB) reporter macrophages using fluidic force microscopy and a micron-scale probe. We describe steps for quantifying the dose of agonist by modeling their diffusion with experimental inputs. We then detail procedures for quantifying and categorizing macrophage responses to individual and varied doses and combining agonist concentration and macrophage response to analyze the NF-κB response to localized TLR stimulation. For complete details on the use and execution of this protocol, please refer to Mulder et al. (2024).1.

Evidence of collective influence in innate sensing using fluidic force microscopy.

The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.

Understanding How Cationic Polymers' Properties Inform Toxic or Immunogenic Responses via Parametric Analysis.

Cationic polymers are widely used materials in diverse biotechnologies. Subtle variations in these polymers' properties can change them from exceptional delivery agents to toxic inflammatory hazards. Conventional screening strategies optimize for function in a specific application rather than observing how underlying polymer-cell interactions emerge from polymers' properties. An alternative approach is to map basic underlying responses, such as immunogenicity or toxicity, as a function of basic physicochemical parameters to inform the design of materials for a breadth of applications. To demonstrate the potential of this approach, we synthesized 107 polymers varied in charge, hydrophobicity, and molecular weight. We then screened this library for cytotoxic behavior and immunogenic responses to map how these physicochemical properties inform polymer-cell interactions. We identify three compositional regions of interest and use confocal microscopy to uncover the mechanisms behind the observed responses. Finally, immunogenic activity is confirmed in vivo. Highly cationic polymers disrupted the cellular plasma membrane to induce a toxic phenotype, while high molecular weight, hydrophobic polymers were uptaken by active transport to induce NLRP3 inflammasome activation, an immunogenic phenotype. Tertiary amine- and triethylene glycol-containing polymers did not invoke immunogenic or toxic responses. The framework described herein allows for the systematic characterization of new cationic materials with different physicochemical properties for applications ranging from drug and gene delivery to antimicrobial coatings and tissue scaffolds.

Nanovaccine that activates the NLRP3 inflammasome enhances tumor specific activation of anti-cancer immunity.

Neoantigen cancer vaccines that target tumor specific mutations are emerging as a promising modality for cancer immunotherapy. To date, various approaches have been adopted to enhance efficacy of these therapies, but the low immunogenicity of neoantigens has hindered clinical application. To address this challenge, we developed a polymeric nanovaccine platform that activates the NLRP3 inflammasome, a key immunological signaling pathway in pathogen recognition and clearance. The nanovaccine is comprised of a poly (orthoester) scaffold engrafted with a small-molecule TLR7/8 agonist and an endosomal escape peptide that facilitates lysosomal rupture and NLRP3 inflammasome activation. Upon solvent transfer, the polymer self-assembles with neoantigens to form ∼50 nm nanoparticles that facilitate co-delivery to antigen-presenting cells. This polymeric activator of the inflammasome (PAI) was found to induce potent antigen-specific CD8+ T cell responses characterized by IFN-γ and GranzymeB secretion. Moreover, in combination with immune checkpoint blockade therapy, the nanovaccine stimulated robust anti-tumor immune responses against established tumors in EG.7-OVA, B16·F10, and CT-26 models. Results from our studies indicate that NLRP3 inflammasome activating nanovaccines demonstrate promise for development as a robust platform to enhance immunogenicity of neoantigen therapies.

A longitudinal investigation of children internationally adopted at school age.

Most existing research on children adopted internationally has focused on those adopted as infants and toddlers. The current study longitudinally tracked several outcomes, including cognitive, behavioral, emotional, attachment, and family functioning, in 25 children who had been internationally adopted at school age (M = 7.7 years old at adoption, SD = 3.4, range = 4­15 years). We examined the incidence of clinically significant impairments, significant change in outcomes over the three study points, and variables that predicted outcomes over time. Clinically significant impairments in sustained attention, full-scale intelligence, reading, language, executive functioning, externalizing problems, and parenting stress were common, with language and executive functioning impairments present at higher levels in the current study compared with past research focusing on children adopted as infants and toddlers. Over the three study points, significant improvements across most cognitive areas and attachment functioning were observed, though significant worsening in executive functioning and internalizing problems was present. Adoptive family-specific variables, such as greater maternal education, smaller family size, a parenting approach that encouraged age-expected behaviors, home schooling, and being the sole adopted child in the family were associated with greater improvement across several cognitive outcomes. In contrast, decreased parenting stress was predicted by having multiple adopted children and smaller family sizes were associated with greater difficulties with executive functioning. Child-specific variables were also linked to outcomes, with girls displaying worse attachment and poorer cognitive performance and with less time in orphanage care resulting in greater adoption success. Implications for future research and clinical applications are discussed.

A longitudinal investigation of children internationally adopted at school age.

Most existing research on children adopted internationally has focused on those adopted as infants and toddlers. The current study longitudinally tracked several outcomes, including cognitive, behavioral, emotional, attachment, and family functioning, in 25 children who had been internationally adopted at school age (M = 7.7 years old at adoption, SD = 3.4, range = 4-15 years). We examined the incidence of clinically significant impairments, significant change in outcomes over the three study points, and variables that predicted outcomes over time. Clinically significant impairments in sustained attention, full-scale intelligence, reading, language, executive functioning, externalizing problems, and parenting stress were common, with language and executive functioning impairments present at higher levels in the current study compared with past research focusing on children adopted as infants and toddlers. Over the three study points, significant improvements across most cognitive areas and attachment functioning were observed, though significant worsening in executive functioning and internalizing problems was present. Adoptive family-specific variables, such as greater maternal education, smaller family size, a parenting approach that encouraged age-expected behaviors, home schooling, and being the sole adopted child in the family were associated with greater improvement across several cognitive outcomes. In contrast, decreased parenting stress was predicted by having multiple adopted children and smaller family sizes were associated with greater difficulties with executive functioning. Child-specific variables were also linked to outcomes, with girls displaying worse attachment and poorer cognitive performance and with less time in orphanage care resulting in greater adoption success. Implications for future research and clinical applications are discussed.