Abstract Introduction TGFβ−secreting, CD206+ profibrotic macrophages (macs) are key chronic graft-versus-host disease (cGVHD) drivers. Global depletion of macs could compromise protective immunity; therefore, we targeted CD206 + (mannose receptor) macs with mannosylated albumin nanoparticles (MANPs) containing TGFβ siRNA or non-targeting (NT) siRNA for control. A published bleomycin pulmonary fibrosis model showed disease prevention post TGFβ-MANPs internalization by CD206+ monocyte-derived alveolar macs. CD206+ TGFβ+ macs are also critical in sclerodermatous (Scl) GVHD (B10D2>Balb/c). We show that mice with established multiorgan cGVHD/bronchiolitis obliterans (BO) exhibit a progressive increase in lung tissue profibrotic macs (d49 vs. d28) highlighting their pathogenic role. Methods In our cGVHD/BO model, B10.BR recipients receive B6 bone marrow (BM) and a low dose of T cells (control = BM only). Co-culture of CD206 + (M2-like) and CD206neg (M1-like) macs confirmed that MANPs were preferentially internalized by CD206+ macs and effectively reduced TGFβ protein and mRNA with no effect on CD206neg macs. In vivo, MANPs are delivered intravenously 2x/week starting at d28 post transplantation. Results Compared to BM only controls, d49 cGVHD/BO mice had a significant increase (1.5-fold) in lung CD206+ macs frequency (p = 0.0010) and these cells had a 2.5-fold increase in TGFβ expression (p = 0.0070) by flow cytometry fluorescent intensity. TGFβ siRNA vs NT siRNA—loaded MANPs delivered i.v. to cGVHD/BO mice improved lung function to levels comparable to BM controls (n = 10/group): resistance (p < 0.0001), elastance (p < 0.05), compliance (p < 0.05). Early studies using MANP-based delivery of TGFβ siRNA in Scl GVHD improved skin scores (p < 0.0001) compared to NT siRNA delivery. Further studies with MANPs in Scl GVHD are ongoing. Conclusion These results identify CD206+ macs as a tractable therapeutic target with high selectivity and highlight MANP-based siRNA delivery as a promising strategy to limit cGVHD-driven pulmonary fibrosis. Funding Source NIH P01 HL158505 Topic Categories Transplantation Immunology (TRAN)

Pulmonary fibrosis is a progressive, severe respiratory disease, often considered terminal, with a typical life expectancy of only a few years. It is marked by excessive deposition of extracellular matrix proteins, driven by a complex interplay of profibrotic signaling pathways, including contributions from monocyte-derived alveolar macrophages (Mo-AMs) and various immune and stromal cells. In this study, we present a peptide-mannan conjugate nanoparticle (PMNP) platform for the targeted delivery of transforming growth factor-β small interfering RNA (TGF-β siRNA) aimed at halting and reversing pulmonary fibrosis. The nanoparticles of TGF-β siRNA and peptide-mannan conjugates, generated through a solvent-free and easily scalable process, were administered intranasally to specifically target the alveolar macrophage population. In fibrotic models, these nanoparticles effectively reduced Mo-AM infiltration, reprogrammed the macrophage phenotype, and significantly reduced collagen deposition. Our findings suggest that intranasal delivery of TGF-β siRNA via PMNP offers a promising, easily self-assembled, and patient-friendly therapeutic approach for the treatment of lung fibrosis.

Ischemia reperfusion (IR)-induced oxidative stress and inflammation contribute to morbidity and mortality of acute coronary syndrome. Ischemia results in profound hypoxia and tissue dysfunction and subsequent reperfusion further aggravates ischemic cardiac tissue damage. In cardiac IR injury, neutrophils are involved both in causing cardiomyocyte death and in preserving heart tissue homeostasis. We tested the hypothesis that neutrophil subpopulations show distinct functions in the pathogenesis of cardiac IR injury and that their functional heterogeneity can be exploited in subset-specific pharmacological intervention to prevent IR-induced myocardial tissue damage and functional deterioration. Cardiac IR-injury in a mouse model was characterized by the presence of two distinct heart-inflammatory subsets of neutrophils, one that specifically endocytosed albumin nanoparticles (ANPhigh) and one that endocytose few or no ANP (ANPlow). The two subsets had very distinct inflammatory phenotypes. ANPhigh neutrophils expressed significantly greater amounts of inflammatory mediators, such as Il-1b and Ccl3, than ANPlow neutrophils. Targeting the Spleen tyrosine kinase (Syk) specifically in ANPhigh neutrophils post IR reduced cardiac neutrophilic and mononuclear inflammation and drastically decreased infarct size, and prevented the deterioration of cardiac function. Targeting the Syk pathway specifically in a defined subset of neutrophils is a feasible therapy for cardiac IR injury.

Dysregulated neutrophil infiltration and activity in lung tissues are pathogenic features of sepsis-induced acute respiratory distress syndrome (ARDS), an important cause of morbidity and mortality in critically ill patients. Therapeutic reduction of excessive neutrophil transmigration from the vasculature into the distal airspaces to mitigate lung tissue damage and improve survival is an important objective. Human peripheral blood polymorphonuclear neutrophils (PMNs) can be separated into two subsets as recently described: one that readily endocytoses albumin nanoparticles (ANPs) referred to as ANP(high) PMN and another that fails to endocytose ANPs referred to as ANP(low) PMN. Here, we tested the hypothesis that targeting ANP(high) human PMNs with ANPs loaded with the drug piceatannol (PANPs), a selective spleen tyrosine kinase (Syk) inhibitor that inhibits beta 2-integrin signaling, would mitigate lipopolysaccharide (LPS)-induced accumulation of PMNs in human lungs and reduce lung edema. In the present study, human lungs were perfused ex vivo with fresh whole blood containing human PMNs and 6 mg of LPS to induce injury accompanied by PMN infiltration into the lungs. Following the LPS exposure, intravenous injection of PANPs reduced transmigration and accumulation of PMNs in the lungs as determined by bronchoalveolar lavage counts and histologic scoring. A parallel reduction in lung weight gain, a surrogate for pulmonary edema fluid accumulation, suggested protection from edema in this human lung injury model. The results demonstrate the potential of therapeutic drug loading of albumin nanoparticles (PANPs) for targeting an injurious, inflammation-inducing population of PMNs in ARDS patients.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils in vivo, we found two subsets of neutrophils, one that readily endocytosed ANP (ANPhigh neutrophils) and another that failed to endocytose ANP (ANPlow population). These ANPhigh and ANPlow subsets existed side by side simultaneously in bone marrow, peripheral blood, spleen, and lungs, both under basal conditions and after inflammatory challenge. Human peripheral blood neutrophils showed a similar duality. ANPhigh and ANPlow neutrophils had distinct cell surface marker expression and transcriptomic profiles, both in naive mice and in mice after endotoxemic challenge. ANPhigh and ANPlow neutrophils were functionally distinct in their capacities to kill bacteria and to produce inflammatory mediators. ANPhigh neutrophils produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines. Targeting this subset with ANP loaded with the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, mitigated the effects of polymicrobial sepsis by reducing tissue inflammation while fully preserving neutrophilic host-defense function.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)β1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFβ1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFβ-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

Journal Article Multi-Signal Characterization of Biological Structures at Low-Voltage Using STEM-in-SEM Get access Kelly Parker, Kelly Parker Department of Materials Science and Engineering, Northwestern University, Evanston, IL, United States Search for other works by this author on: Oxford Academic Google Scholar Abhalaxmi Singh, Abhalaxmi Singh Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, United States Search for other works by this author on: Oxford Academic Google Scholar Vinayak P Dravid Vinayak P Dravid Department of Materials Science and Engineering, Northwestern University, Evanston, IL, United StatesNorthwestern University Atomic and Nanoscale Characterization Experimental (NUANCE) Center, Northwestern University, Evanston, IL, United States Corresponding author: v-dravid@northwestern.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1108–1110, https://doi.org/10.1017/S1431927622004688 Published: 01 August 2022

Immune memory has been well-established in adaptive immunity but recent studies suggest a key for memory in innate immunity. However, the precise mechanisms and phenotypes of innate immune memory in macrophages remain poorly understood. Here, we studied the memory responses of lung alveolar macrophages (AMs) exposed consecutively (2 exposures, one week apart) intratracheally to the bacterial endotoxin lipopolysaccharide (LPS). We observed a 60%–80% loss of AMs following the initial LPS exposure, consistent with the known cell death of resident AMs during severe inflammatory injury followed by gradual replenishment. Importantly, AM loss following the second LPS injury was markedly attenuated. Lineage tracing using a CX3CR1-monocyte reporter showed no increase in monocyte-derived AMs, thus suggesting that the higher AM numbers after the second LPS exposure were due to greater resilience of AMs and not due to rapid replenishment by circulating monocytes. RNA-Seq analysis of showed higher expression of pro-survival genes and anti-inflammatory cytokines such as IL-10 in memory AMs (post 1 week LPS) than in naïve AMs. Ex vivo and in vivo functional assay showed that these memory AMs demonstrated enhanced efferocytosis of cellular debris, consistent with a pro-resolving phenotype. We also found upregulation of the transcription factor KLF4 which promotes cell survival as well as pro-resolving macrophage polarization. Our findings suggest that alveolar macrophages are reprogrammed by an initial inflammatory exposure to increase their resilience to pro-apoptotic stimuli and to promote resolution of inflammation. Such a memory mechanism could be leveraged therapeutically in severe inflammatory diseases.

Inflammatory tissue injury such as acute lung injury (ALI) is a disorder that leads to respiratory failure, a major cause of morbidity and mortality worldwide. Excessive neutrophil influx is a critical pathogenic factor in the development of ALI. Here, we identify the subset of neutrophils that is responsible for ALI and lethality in polymicrobial sepsis. The pro-inflammatory neutrophil subpopulation was characterized by its unique ability to endocytose albumin nanoparticles (ANP), upregulation of pro-inflammatory cytokines and chemokines as well as the excessive production of reactive oxygen species (ROS) in models of endotoxemia and septicemia. ANP delivery of the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, to the susceptible subset of neutrophils, prevented ALI and mortality in mice subjected to polymicrobial infection. Targeted inhibition of Syk in ANP-susceptible neutrophils had no detrimental effect on neutrophil-dependent host defense because the subset of ANP low neutrophils effectively controlled polymicrobial infection. The results show that neutrophil heterogeneity can be leveraged therapeutically to prevent ALI without compromising host defense.

The panorama of cancer treatment has taken a considerable leap over the last decade with the advancement in the upcoming novel therapies combined with modern diagnostics. Nanotheranostics is an emerging science that holds tremendous potential as a contrivance by integrating therapy and imaging in a single probe for cancer diagnosis and treatment thus offering the advantage like tumor-specific drug delivery and at the same time reduced side effects to normal tissues. The recent surge in nanomedicine research has also paved the way for multimodal theranostic nanoprobe towards personalized therapy through interaction with a specific biological system. This review presents an overview of the nano theranostics approach in cancer management and a series of different nanomaterials used in theranostics and the possible challenges with future directions.

Targeted cancer therapy facilitates localizing the action of chemotherapeutic drugs at the tumor site enhancing the therapeutic efficacy and reducing the side effects to the healthy cells. The homing property of mesenchymal stem cells (MSCs), towards the tumor tissues makes them a potential cell-based delivery system for targeted cancer therapy. Along with chemotherapy, hyperthermia has gained interest as a treatment modality of cancer due to the higher sensitivity of the cancer cells towards heat and also due to its action on tumor cells to enhance sensitization towards chemotherapy or radiotherapy. In the current study, we have shown the multifaceted application of magnetic nanoparticles (MNPs) as a drug delivery vehicle to deliver anti-cancer drug paclitaxel and also as an inducer for magnetic hyperthermia under alternating magnetic field. The combined approach of paclitaxel loaded MNPs and hyperthermia demonstrated enhanced therapeutic efficacy as compared to any single therapy. Further, we have employed MSCs as carrier for these drugs loaded MNPs to achieve targeted and uniform distribution of the MNPs at the tumor site. We have evaluated the efficacy of the system in in vitro and in vivo prostate tumor model. The in vivo tumor study shows uniform distribution of drug loaded MNPs with use of mesenchymal stem cells as a delivery vehicle and combination of hyperthermia and MNP mediated drug delivery results in better tumor remission.

B-cell lymphoma cells depend upon cholesterol to maintain pro-proliferation and pro-survival signaling via the B-cell receptor. Targeted cholesterol depletion of lymphoma cells is an attractive therapeutic strategy. We report here high-density lipoprotein mimicking magnetic nanostructures (HDL-MNSs) that can bind to the high-affinity HDL receptor, scavenger receptor type B1 (SR-B1), and interfere with cholesterol flux mechanisms in SR-B1 receptor positive lymphoma cells, causing cellular cholesterol depletion. In addition, the MNS core can be utilized for its ability to generate heat under an external radio frequency field. The thermal activation of MNS can lead to both innate and adaptive antitumor immune responses by inducing the expression of heat shock proteins that lead to activation of antigen presenting cells and finally lymphocyte trafficking. In the present study, we demonstrate SR-B1 receptor mediated binding and cellular uptake of HDL-MNS and prevention of phagolysosome formation by transmission electron microscopy, fluorescence microscopy, and ICP-MS analysis. The combinational therapeutics of cholesterol depletion and thermal activation significantly improves therapeutic efficacy in SR-B1 expressing lymphoma cells. HDL-MNS reduces the T2 relaxation time under magnetic resonance imaging (MRI) more effectively compared with a commercially available contrast agent, and the specificity of HDL-MNS toward the SR-B1 receptor leads to differential contrast between SR-B1 positive and negative cells suggesting its utility in diagnostic imaging. Overall, we have demonstrated that HDL-MNSs have cell specific targeting efficiency, can modulate cholesterol efflux, can induce thermal activation mediated antitumor immune response, and possess high contrast under MRI, making it a promising theranostic platform in lymphoma.

We report magnetic nanostructure-stabilized lipid nanocapsules (MLNCs) that show superior structural stability and theranostic properties compared to conventional lipid-based nanocarriers. As therapeutic nanocarriers, the MLNCs exhibit a therapeutic efficacy that is 16 times greater than that of free drugs due to their high payload capacity and actuated drug release ability. In addition, the magnetic resonance contrast enhancement of the MLNCs is nine times higher than that of a clinically approved T2 MRI contrast agent (ferumoxytol), demonstrating the diagnostic imaging capability of the MLNCs in MRI. The self-assembly method to synthesize the lipid nanocapsules is extended to other types of nanoparticles (gold nanoparticles and quantum dots) to produce lipid nanohybrids with distinct physical properties.

Mesenchymal stem cells (MSCs) have gained much interest to be used as targeting vehicle in cancer therapy due to the intrinsic tumor-homing behavior associated with them. In this scenario, superparamagnetic nanoparticles are emerging as an ideal probe for noninvasive cell tracking for different stem cell applications. In the study, it is demonstrated that the formulated aqueous dispersible glyceryl monooleate coated magnetic nanoparticles (MNPs) can act as a better labeling and efficient tracking agent without affecting the inherent properties of MSCs. The MNPs-MSCs facilitate the stem cell tracking by magnetic resonance imaging at a very low cell number having high T-2 relaxivity and potentiates the use of MNPs-MSCs as a prospective diagnostic tool. Most importantly, the homing of MNPs-MSCs toward inflammation site, subcutaneous prostate tumor (small as well as large tumor), and in orthotopic prostate tumor suggests the clinical relevance of the system. In addition, intraperitoneal delivery of MNPs-MSCs shows enhanced tumor accumulation and less sequestration in liver as revealed by in vivo imaging and histological studies. The results here demonstrate that MNPs-MSCs may prove as a better targeted delivery agent for early diagnosis of tumors even of smaller size.