Nanoparticle dose and antigen loading attenuate antigen-specific T-cell responses.

Immune-mediated hypersensitivities such as autoimmunity, allergy, and allogeneic graft rejection are treated with therapeutics that suppress the immune system, and the lack of specificity is associated with significant side effects. The delivery of disease-relevant antigens (Ags) by carrier systems such as poly(lactide-co-glycolide) nanoparticles (PLG-Ag) and carbodiimide (ECDI)-fixed splenocytes (SP-Ag) has demonstrated Ag-specific tolerance induction in model systems of these diseases. Despite therapeutic outcomes by both platforms, tolerance is conferred with different efficacy. This investigation evaluated Ag loading and total particle dose of PLG-Ag on Ag presentation in a coculture system of dendritic cells (DCs) and Ag-restricted T cells, with SP-Ag employed as a control. CD25 expression was observed in nearly all T cells even at low concentrations of PLG-Ag, indicating efficient presentation of Ag by dendritic cells. However, the secretion of IL-2, Th1, and Th2 cytokines (IFNγ and IL-4, respectively) varied depending on PLG-Ag concentration and Ag loading. Concentration escalation of soluble Ag resulted in an increase in IL-2 and IFNγ and a decrease in IL-4. Treatment with PLG-Ag followed a similar trend but with lower levels of IL-2 and IFNγ secreted. Transcriptional Activity CEll ARrays (TRACER) were employed to measure the real-time transcription factor (TF) activity in Ag-presenting DCs. The kinetics and magnitude of TF activity was dependent on the Ag delivery method, concentration, and Ag loading. Ag positively regulated IRF1 activity and, as carriers, NPs and ECDI-treated SP negatively regulated this signaling. The effect of Ag loading and dose on tolerance induction were corroborated in vivo using the delayed-type hypersensitivity (DTH) and experimental autoimmune encephalomyelitis (EAE) mouse models where a threshold of 8 µg/mg Ag loading and 0.5 mg PLG-Ag dose were required for tolerance. Together, the effect of Ag loading and dosing on in vitro and in vivo immune regulation provide useful insights for translating Ag-carrier systems for the clinical treatment of immune disorders.

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer.

BACKGROUND: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. METHODS: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. RESULTS: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). CONCLUSION: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

Local Immunomodulation with Anti-inflammatory Cytokine-Encoding Lentivirus Enhances Functional Recovery after Spinal Cord Injury.

Trauma to the spinal cord and associated secondary inflammation can lead to permanent loss of sensory and motor function below the injury level, with the resulting environment serving as a barrier that limits regeneration. In this study, we investigate the localized expression of anti-inflammatory cytokines IL-10 and IL-4 via lentiviral transduction in multichannel bridges. Porous multichannel bridges provide physical guidance for axonal outgrowth with the cytokines hypothesized to modulate the neuroinflammatory microenvironment and enhance axonal regeneration. Gene expression analyses indicated that induced IL-10 and IL-4 expression decreased expression of pro-inflammatory genes and increased pro-regenerative genes relative to control. Moreover, these factors led to increased numbers of axons and myelination, with approximately 45% of axons myelinated and the number of oligodendrocyte myelinated axons significantly increased by 3- to 4-fold. Furthermore, the combination of a bridge with IL-10 and IL-4 expression improved locomotor function after injury to an average score of 6 relative to an average score of 3 for injury alone. Collectively, these studies highlight the potential for localized immunomodulation to decrease secondary inflammation and enhance regeneration that may have numerous applications.

Dynamic microRNA activity identifies therapeutic targets in trastuzumab-resistant HER2+ breast cancer.

MicroRNAs (miRNAs) are implicated in numerous physiologic and pathologic processes, such as the development of resistance to chemotherapy. Determining the role of miRNAs in these processes is often accomplished through measuring miRNA abundance by polymerase chain reaction, sequencing, or microarrays. We have developed a system for the large-scale monitoring of dynamic miRNA activity and have applied this system to identify the contribution miRNA activity to the development of trastuzumab resistance in a cell model of HER2+ breast cancer. MiRNA activity measurements identified significantly different activity levels between BT474 cells (HER2 + breast cancer) and BT474R cells (HER2 + breast cancer cells selected for resistance to trastuzumab). We created a library of 32 miRNA reporter constructs, which were delivered by lentiviral transduction into cells, and miRNA activity was quantified by bioluminescence imaging. Upon treatment with the bioimmune therapy, trastuzumab, the activity of 11 miRNAs were significantly altered in parental BT474 cells, and 20 miRNAs had significantly altered activity in the therapy-resistant BT474R cell line. A combination of statistical, network and classification analysis was applied to the dynamic data, which identified miR-21 as a controlling factor in trastuzumab response. Our data suggested downregulation of miR-21 activity was associated with resistance, which was confirmed in an additional HER2 + breast cancer cell line, SKBR3. Collectively, the dynamic miRNA activity measurements and analysis provided a system to identify new potential therapeutic targets in treatment-resistant cancers.

Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) culture models of chondrogenic ATDC5 cells, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Both culture models were treated with ITS+, 10mM ß-glycerophosphate (ßGP), or ITS+/10mM ßGP, which resulted in three levels of Pi abundance in cultures: basal (Pi/DNA <10ng/µg), moderate (Pi/DNA=25.3 - 32.3ng/µg), and high abundance (Pi/DNA >60ng/µg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM ßGP resulted in moderate abundance in 2D and high abundance in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters reduced phosphate-induced expression of chondrogenic markers. When 3D ITS+/ßGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

Systems analysis of dynamic transcription factor activity identifies targets for treatment in Olaparib resistant cancer cells.

The development of resistance to targeted therapeutics is a challenging issue for the treatment of cancer. Cancers that have mutations in BRCA, a DNA repair protein, have been treated with poly(ADP-ribose) polymerase (PARP) inhibitors, which target a second DNA repair mechanism with the aim of inducing synthetic lethality. While these inhibitors have shown promise clinically, the development of resistance can limit their effectiveness as a therapy. This study investigated mechanisms of resistance in BRCA-mutated cancer cells (HCC1937) to Olaparib (AZD2281) using TRACER, a technique for measuring dynamics of transcription factor (TF) activity in living cells. TF activity was monitored in the parental HCC1937 cell line and two distinct resistant cell lines, one with restored wild-type BRCA1 and one with acquired resistance independent of BRCA1 for 48 h during treatment with Olaparib. Partial least squares discriminant analysis (PLSDA) was used to categorize the three cell types based on TF activity, and network analysis was used to investigate the mechanism of early response to Olaparib in the study cells. NOTCH signaling was identified as a common pathway linked to resistance in both Olaparib-resistant cell types. Western blotting confirmed upregulation of NOTCH protein, and sensitivity to Olaparib was restored through co-treatment with a gamma secretase inhibitor. The identification of NOTCH signaling as a common pathway contributing to PARP inhibitor resistance by TRACER indicates the efficacy of transcription factor dynamics in identifying targets for intervention in treatment-resistant cancer and provides a new method for determining effective strategies for directed chemotherapy. Biotechnol. Bioeng. 2017;114: 2085-2095. © 2017 Wiley Periodicals, Inc.

Engineered antifouling microtopographies: surface pattern effects on cell distribution.

Microtopography has been observed to lead to altered attachment behavior for marine fouling organisms; however, quantification of this phenomenon is lacking in the scientific literature. Here, we present quantitative measurement of the disruption of normal attachment behavior of the fouling algae Ulva linza by antifouling microtopographies. The distribution of the diatom Navicula incerta was shown to be unaffected by the presence of topography. The radial distribution function was calculated for both individual zoospores and cells as well as aggregates of zoospores from attachment data for a variety topographic configurations and at a number of different attachment densities. Additionally, the screening distance and maximum values were mapped according to the location of zoospore aggregates within a single unit cell. We found that engineered topographies decreased the distance between spore aggregates compared to that for a smooth control surface; however, the distributions for individual spores were unchanged. We also found that the local attachment site geometry affected the screening distance for aggregates of zoospores, with certain geometries decreasing screening distance and others having no measurable effect. The distribution mapping techniques developed and explored in this article have yielded important insight into the design parameters for antifouling microtopographies that can be implemented in the next generation of antifouling surfaces.

Human Breast Cancer Cell Lines Differentially Modulate Signaling from Distant Microenvironments, Which Reflects Their Metastatic Potential.

Metastasis is the stage at which the prognosis substantially decreases for many types of cancer. The ability of tumor cells to metastasize is dependent upon the characteristics of the tumor cells, and the conditioning of distant tissues that support colonization by metastatic cells. In this report, we investigated the systemic alterations in distant tissues caused by multiple human breast cancer cell lines and the impact of these alterations on the tumor cell phenotype. We observed that the niche within the lung, a common metastatic site, was significantly altered by MDA-MB-231, MCF7, and T47 tumors, and that the lung microenvironment stimulated, to differing extents, an epithelial-to-mesenchymal transition (EMT), reducing proliferation, increasing transendothelial migration and senescence, with no significant impact on cell death. We also investigated the ability of an implantable scaffold, which supports the formation of a distant tissue, to serve as a surrogate for the lung to identify systemic alterations. The scaffolds are conditioned by the primary tumor similarly to the lung for each tumor type, evidenced by promoting a pro-EMT profile. Collectively, we demonstrate that metastatic and non-metastatic breast cancers condition distant tissues, with distinct effects on tumor cell responses, and that a surrogate tissue can distinguish the metastatic potential of human breast cancer cell lines in an accessible site that avoids biopsy of a vital organ.

Engineered antifouling microtopographies: an energetic model that predicts cell attachment.

We have developed a model for the prediction of cell attachment to engineered microtopographies based on two previous models: the attachment point theory and the engineered roughness index (ERI) model. The new surface energetic attachment (SEA) model is based on both the properties of the cell-material interface and the size and configuration of the topography relative to the organism. We have used Monte Carlo simulation to examine the SEA model's ability to predict relative attachment of the green alga Ulva linza to different locations within a unit cell. We have also compared the predicted relative attachment for Ulva linza, the diatom Navicula incerta, the marine bacterium Cobetia marina, and the barnacle cyprid Balanus amphitrite to a wide variety of microtopographies. We demonstrate good correlation between the experimental results and the model results for all tested experimental data and thus show the SEA model may be used as a powerful indicator of the efficacy for antifouling topographies.

A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs.

Biomaterial scaffolds mimicking the environment in metastatic organs can deconstruct complex signals and facilitate the study of cancer progression and metastasis. Here we report that a subcutaneous scaffold implant in mouse models of metastatic breast cancer in female mice recruits lung-tropic circulating tumor cells yet suppresses their growth through potent in situ antitumor immunity. In contrast, the lung, the endogenous metastatic organ for these models, develops lethal metastases in aggressive breast cancer, with less aggressive tumor models developing dormant lungs suppressing tumor growth. Our study reveals multifaceted roles of neutrophils in regulating metastasis. Breast cancer-educated neutrophils infiltrate the scaffold implants and lungs, secreting the same signal to attract lung-tropic circulating tumor cells. Second, antitumor and pro-tumor neutrophils are selectively recruited to the dormant scaffolds and lungs, respectively, responding to distinct groups of chemoattractants to establish activated or suppressive immune environments that direct different fates of cancer cells.

Human Lung Organoid Culture in Alginate With and Without Matrigel to Model Development and Disease.

Human lung organoids (HLOs) are enabling the study of human lung development and disease by modeling native organ tissue structure, cellular composition, and cellular organization. In this report, we demonstrate that HLOs derived from human pluripotent stem cells cultured in alginate, a fully defined nonanimal product substrate, exhibit enhanced cellular differentiation compared with HLOs cultured in the commercially available Matrigel. More specifically, we observed an earlier onset and increase in the number of multiciliated cells, along with mucus producing MUC5AC+ goblet-like cells that were not observed in HLOs cultured in Matrigel. The epithelium in alginate-grown HLOs was organized in a pseudostratified epithelium with airway basal cells lining the basal lamina, but with the apical surface of cells on the exterior of the organoid. We further observed that HLOs cultured in Matrigel exhibited mesenchymal overgrowth that was not present in alginate cultures. The containment of the mesenchyme within HLOs in alginate enabled modeling of key features of idiopathic pulmonary fibrosis (IPF) by treatment with transforming growth factor ß (TGFß). TGFß treatment resulted in morphological changes including an increase in mesenchymal growth, increased expression of IPF markers, and decreased numbers of alveolar-like cells. This culture system provides a model to study the interaction of the mesenchyme with the epithelium during lung development and diseased states such as IPF.

Single-cell RNA-sequencing identifies anti-cancer immune phenotypes in the early lung metastatic niche during breast cancer.

Microenvironmental changes in the early metastatic niche may be exploited to identify therapeutic targets to inhibit secondary tumor formation and improve disease outcomes. We dissected the developing lung metastatic niche in a model of metastatic, triple-negative breast cancer using single-cell RNA-sequencing. Lungs were extracted from mice at 7-, 14-, or 21 days after tumor inoculation corresponding to the pre-metastatic, micro-metastatic, and metastatic niche, respectively. The progression of the metastatic niche was marked by an increase in neutrophil infiltration (5% of cells at day 0 to 81% of cells at day 21) and signaling pathways corresponding to the hallmarks of cancer. Importantly, the pre-metastatic and early metastatic niche were composed of immune cells with an anti-cancer phenotype not traditionally associated with metastatic disease. As expected, the metastatic niche exhibited pro-cancer phenotypes. The transition from anti-cancer to pro-cancer phenotypes was directly associated with neutrophil and monocyte behaviors at these time points. Predicted metabolic, transcription factor, and receptor-ligand signaling suggested that changes in the neutrophils likely induced the transitions in the other immune cells. Conditioned medium generated by cells extracted from the pre-metastatic niche successfully inhibited tumor cell proliferation and migration in vitro and the in vivo depletion of pre-metastatic neutrophils and monocytes worsened survival outcomes, thus validating the anti-cancer phenotype of the developing niche. Genes associated with the early anti-cancer response could act as biomarkers that could serve as targets for the treatment of early metastatic disease. Such therapies have the potential to revolutionize clinical outcomes in metastatic breast cancer.

Implications of TGFß Signaling and CDK Inhibition for the Treatment of Breast Cancer.

TGFß signaling enacts tumor-suppressive functions in normal cells through promotion of several cell regulatory actions including cell-cycle control and apoptosis. Canonical TGFß signaling proceeds through phosphorylation of the transcription factor, SMAD3, at the C-terminus of the protein. During oncogenic progression, this tumor suppressant phosphorylation of SMAD3 can be inhibited. Overexpression of cyclins D and E, and subsequent hyperactivation of cyclin-dependent kinases 2/4 (CDKs), are often observed in breast cancer, and have been associated with poor prognosis. The noncanonical phosphorylation of SMAD3 by CDKs 2 and 4 leads to the inhibition of tumor-suppressive function of SMAD3. As a result, CDK overactivation drives oncogenic progression, and can be targeted to improve clinical outcomes. This review focuses on breast cancer, and highlights advances in the understanding of CDK-mediated noncanonical SMAD3 phosphorylation. Specifically, the role of aberrant TGFß signaling in oncogenic progression and treatment response will be examined to illustrate the potential for therapeutic discovery in the context of cyclins/CDKs and SMAD3.

Towards systems tissue engineering: Elucidating the dynamics, spatial coordination, and individual cells driving emergent behaviors.

Biomaterial systems have enabled the in vitro production of complex, emergent tissue behaviors that were not possible with conventional two-dimensional culture systems, allowing for analysis of both normal development and disease processes. We propose that the path towards developing the design parameters for biomaterial systems lies with identifying the molecular drivers of emergent behavior through leveraging technological advances in systems biology, including single cell omics, genetic engineering, and high content imaging. This growing research opportunity at the intersection of the fields of tissue engineering and systems biology - systems tissue engineering - can uniquely interrogate the mechanisms by which complex tissue behaviors emerge with the potential to capture the contribution of i) dynamic regulation of tissue development and dysregulation, ii) single cell heterogeneity and the function of rare cell types, and iii) the spatial distribution and structure of individual cells and cell types within a tissue. By leveraging advances in both biological and materials data science, systems tissue engineering can facilitate the identification of biomaterial design parameters that will accelerate basic science discovery and translation.

Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+ breast cancer.

The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-ß signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.

Adrenergic Blockade Promotes Maintenance of Dormancy in Prostate Cancer Through Upregulation of GAS6.

Men diagnosed with localized prostate cancer can develop metastases many years after initial treatment, resulting in a poor prognosis. The purpose of this study was to investigate the mechanisms by which signaling through norepinephrine (NE) may incite relapse of quiescent prostate cancer. We used an unbiased bioinformatics pipeline to examine mechanisms for recurrence related to sympathetic signaling in the bone marrow. A transcription factor cell array identified ATF1, RAR, and E2F as key nodes in prostate cancer cells exiting quiescence through adrenergic signaling. Subsequent secretome analysis identified GAS6 as affecting activity of these three factors, leading to cell cycle reentry. GAS6 expression was downregulated in osteoblasts through activation of the cAMP pathway and was targeted in vitro and in vivo using pharmacological agents (propranolol and phentolamine). Propranolol increased expression of GAS6 by osteoblasts, and phentolamine significantly inhibited expression. Propranolol treatment was sufficient to both increase GAS6 expression in marrow osteoblasts as well as eliminate the effects of NE signaling on GAS6 expression. These results demonstrate a strong correlation between adrenergic signaling, GAS6 expression, and recurrence in prostate cancer, suggesting a novel therapeutic direction for patients at high risk of metastasis.

Metastatic Conditioning of Myeloid Cells at a Subcutaneous Synthetic Niche Reflects Disease Progression and Predicts Therapeutic Outcomes.

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease. SIGNIFICANCE: Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/602/F1.large.jpg.See related commentary by Wolf and Elisseeff, p. 377.

Cargo-less nanoparticles program innate immune cell responses to toll-like receptor activation.

Developing biomaterials to control the responsiveness of innate immune cells represents a clinically relevant approach to treat diseases with an underlying inflammatory basis, such as sepsis. Sepsis can involve activation of Toll-like receptor (TLR) signaling, which activates numerous inflammatory pathways. The breadth of this inflammation has limited the efficacy of pharmacological interventions that target a single molecular pathway. Here, we developed cargo-less particles as a single-agent, multi-target platform to elicit broad anti-inflammatory action against innate immune cells challenged by multiple TLR agonists. The particles, prepared from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA), displayed potent molecular weight-, polymer composition-, and charge-dependent immunomodulatory properties, including downregulation of TLR-induced costimulatory molecule expression and cytokine secretion. Particles prepared using the anionic surfactant poly(ethylene-alt-maleic acid) (PEMA) significantly blunted the responses of antigen presenting cells to TLR4 (lipopolysaccharide) and TLR9 (CpG-ODN) agonists, demonstrating broad inhibitory activity to both extracellular and intracellular TLR ligands. Interestingly, particles prepared using poly(vinyl alcohol) (PVA), a neutrally-charged surfactant, only marginally inhibited inflammatory cytokine secretions. The biochemical pathways modulated by particles were investigated using TRanscriptional Activity CEll aRrays (TRACER), which implicated IRF1, STAT1, and AP-1 in the mechanism of action for PLA-PEMA particles. Using an LPS-induced endotoxemia mouse model, administration of PLA-PEMA particles prior to or following a lethal challenge resulted in significantly improved mean survival. Cargo-less particles affect multiple biological pathways involved in the development of inflammatory responses by innate immune cells and represent a potentially promising therapeutic strategy to treat severe inflammation.

Reducing inflammation through delivery of lentivirus encoding for anti-inflammatory cytokines attenuates neuropathic pain after spinal cord injury.

Recently, many clinical trials have challenged the efficacy of current therapeutics for neuropathic pain after spinal cord injury (SCI) due to their life-threatening side-effects including addictions. Growing evidence suggests that persistent inflammatory responses after primary SCI lead to an imbalance between anti-inflammation and pro-inflammation, resulting in pathogenesis and maintenance of neuropathic pain. Conversely, a variety of data suggest that inflammation contributes to regeneration. Herein, we investigated long-term local immunomodulation using anti-inflammatory cytokine IL-10 or IL-4-encoding lentivirus delivered from multichannel bridges. Multichannel bridges provide guidance for axonal outgrowth and act as delivery vehicles. Anti-inflammatory cytokines were hypothesized to modulate the pro-nociceptive inflammatory niche and promote axonal regeneration, leading to neuropathic pain attenuation. Gene expression analyses demonstrated that IL-10 and IL-4 decreased pro-nociceptive genes expression versus control. Moreover, these factors resulted in an increased number of pro-regenerative macrophages and restoration of normal nociceptors expression pattern. Furthermore, the combination of bridges with anti-inflammatory cytokines significantly alleviated both mechanical and thermal hypersensitivity relative to control and promoted axonal regeneration. Collectively, these studies highlight that immunomodulatory strategies target multiple barriers to decrease secondary inflammation and attenuate neuropathic pain after SCI.

Synergy of Paracrine Signaling During Early-Stage Mouse Ovarian Follicle Development In Vitro.

Introduction: Paracrine signals, such as soluble cytokines and extracellular matrix cues, are essential for the survival and development of multicellular ovarian follicles. While it is well established that hydrogel-based culture systems successfully support the growth of late-stage follicles for fertility preservation, growing small, early-stage ovarian follicles still proves to be challenging. We hypothesized that paracrine factors secreted from neighboring follicles may be crucial for improving the survival of early-stage follicles in vitro. Methods: To test our hypothesis, we investigated the bi-directional crosstalk of the paracrine signals, such as cell-secreted cytokines, sex hormones and transcription factors (TFs), in follicles encapsulated and cultured for 12 days in alginate in groups of five (5×) and ten (10×). Results: The differential profiles of TF activity and secretome during folliculogenesis were analyzed using TRanscriptional Activity CEllular aRray (TRACER) and data-driven multivariate modeling approach. The mechano- and oxygen-responsive TFs, NF-κB and HIF1, exhibited a unique upregulation signature in 10× follicles. Consistently, levels of proangiogenic factors, such as VEGF-A and angiopoietin-2, were significantly higher in 10× follicles than those in 5× follicles, reaching 269.77 and 242.82 pg/mL on the last day of culture. The analysis of TRACER and secreted cytokines also revealed critical early interactions between cytokines and TFs, correlating with the observed phenotypical and functional differences between conditions. Conclusions: We identified unique signatures of synergism during successful early-stage ovarian follicle development. These findings bring us closer to understanding of mechanisms underlying the downstream effects of interactions between the extracellular microenvironment and early-stage folliculogenesis in vitro.