The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity.

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.

Enhanced Mott cell formation linked with IgM Fc receptor (FcµR) deficiency.

In previous studies, Mott cells, an unusual form of plasma cells containing Ig-inclusion bodies, were frequently observed in peripheral lymphoid tissues in our IgM Fc receptor (FcµR)-deficient (KO) mouse strain. Because of discrepancies in the reported phenotypes of different Fcmr KO mouse strains, we here examined two additional available mutant strains and confirmed that such enhanced Mott-cell formation was a general phenomenon associated with FcµR deficiency. Splenic B cells from Fcmr KO mice clearly generated more Mott cells than those from WT mice when stimulated in vitro with LPS alone or a B-1, but not B-2, activation cocktail. Nucleotide sequence analysis of the Ig variable regions of a single IgMλ+ Mott-hybridoma clone developed from splenic B-1 B cells of Fcmr KO mice revealed the near (VH) or complete (Vλ) identity with the corresponding germline gene segments and the addition of six or five nucleotides at the VH/DH and DH/JH junctions, respectively. Transduction of an FcµR cDNA into the Mott hybridoma significantly reduced cells containing IgM-inclusion bodies with a concomitant increase in IgM secretion, leading to secreted IgM binding to FcµR expressed on Mott transductants. These findings suggest a regulatory role of FcµR in the formation of Mott cells and IgM-inclusion bodies.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene-Cyanines Are Loud and Photostable NIR Imaging Agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents: which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches to cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (here: >3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Propagation of structured light through tissue-mimicking phantoms.

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.

Induction of microRNA-155 is TLR- and type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis.

Helicobacter pylori is a gastric pathogen responsible for a high disease burden worldwide. Deregulated inflammatory responses, possibly involving macrophages, are implicated in H. pylori-induced pathology, and microRNAs, such as miR-155, have recently emerged as crucial regulators of innate immunity and inflammatory responses. miR-155 is regulated by Toll-like receptor (TLR) ligands in monocyte-derived cells and has been shown to be induced in macrophages during H. pylori infection. Here, we investigated the regulation of miR-155 expression in primary murine bone marrow-derived macrophages (BMMs) during H. pylori infection and examined the downstream mRNA targets of this microRNA using microarray analysis. We report TLR2/4- and NOD1/2-independent up-regulation of miR-155, which was found to be dependent on the major H. pylori pathogenicity determinant, the type IV secretion system (T4SS). miR-155 expression was dependent on NF-κB signaling but was independent of CagA. Microarray analysis identified known gene targets of miR-155 in BMMs during H. pylori infection that are proapoptotic. We also identified and validated miR-155 binding sites in the 3' UTRs of the targets, Tspan14, Lpin1, and Pmaip1. We observed that H. pylori-infected miR-155(-/-) BMMs were significantly more susceptible to cisplatin DNA damage-induced apoptosis than were wild-type BMMs. Thus, our data suggest a function for the prototypical H. pylori pathogenicity factor, the T4SS, in the up-regulation of miR-155 in BMMs. We propose the antiapoptotic effects of miR-155 could enhance macrophage resistance to apoptosis induced by DNA damage during H. pylori infection.

Immune cell imaging using multi-spectral optoacoustic tomography.

Multispectral optoacoustic tomography (MSOT) offers the potential to image in high-resolution cells tagged with optical labels. In contrast to single wavelength imaging, multispectral excitation and spectral unmixing can differentiate labeled moieties over tissue absorption in the absence of background measurements. This feature can enable longitudinal cellular biology studies well beyond the depths reached by optical microscopy. However, the relation between spectrally resolved fluorescently labeled cells and optoacoustic detection has not been systematically investigated. Herein, we measured titrations of fluorescently labeled cells and establish the optoacoustic signal generated by these cells as a function of cell number and across different cell types. We then assess the MSOT sensitivity to resolve cells implanted in animals.

Performance Assessment and Quality Control of Fluorescence Molecular Endoscopy with a Multi-Parametric Rigid Standard.

Fluorescence molecular endoscopy (FME) is emerging as a "red-flag" technique with potential to deliver earlier, faster, and more personalized detection of disease in the gastrointestinal tract, including cancer, and to gain insights into novel drug distribution, dose finding, and response prediction. However, to date, the performance of FME systems is assessed mainly by endoscopists during a procedure, leading to arbitrary, potentially biased, and heavily subjective assessment. This approach significantly affects the repeatability of the procedures and the interpretation or comparison of the acquired data, representing a major bottleneck towards the clinical translation of the technology. Herein, we propose a robust methodology for FME performance assessment and quality control that is based on a novel multi-parametric rigid standard. This standard enables the characterization of an FME system's sensitivity through a single acquisition, performance comparison of multiple systems, and, for the first time, quality control of a system as a function of time and number of usages. We show the photostability of the standard experimentally and demonstrate how it can be used to characterize the performance of an FME system. Moreover, we showcase how the standard can be employed for quality control of a system. In this study, we find that the use of composite fluorescence standards before endoscopic procedures can ensure that an FME system meets the performance criteria and that components prone to performance degradation are replaced in time, avoiding disruption of clinical endoscopy logistics. This will help overcome a major barrier for the translation of FME into the clinics.

Tuning the photophysical properties of cyanine by barbiturate functionalization and nanoformulation for efficient optoacoustics- guided phototherapy.

Cyanine derivatives are organic dyes widely used for optical imaging. However, their potential in longitudinal optoacoustic imaging and photothermal therapy remains limited due to challenges such as poor chemical stability, poor photostability, and low photothermal conversion. In this study, we present a new structural modification for cyanine dyes by introducing a strongly electron-withdrawing group (barbiturate), resulting in a new series of barbiturate-cyanine dyes (BC810, BC885, and BC1010) with suppressed fluorescence and enhanced stability. Furthermore, the introduction of BC1010 into block copolymers (PEG114-b-PCL60) induces aggregation-caused quenching, further boosting the photothermal performance. The photophysical properties of nanoparticles (BC1010-NPs) include their remarkably broad absorption range from 900 to 1200 nm for optoacoustic imaging, allowing imaging applications in NIR-I and NIR-II windows. The combined effect of these strategies, including improved photostability, enhanced nonradiative relaxation, and aggregation-caused quenching, enables the detection of optoacoustic signals with high sensitivity and effective photothermal treatment of in vivo tumor models when BC1010-NPs are administered before irradiation with a 1064 nm laser. This research introduces a barbiturate-functionalized cyanine derivative with optimal properties for efficient optoacoustics-guided theranostic applications. This new compound holds significant potential for biomedical use, facilitating advancements in optoacoustic-guided diagnostic and therapeutic approaches.

Alginate beads as a highly versatile test-sample for optoacoustic imaging.

Test-samples are necessary for the development of emerging imaging approaches such as optoacoustics (OA); these can be used to benchmark new labeling agents and instrumentation, or to characterize image analysis algorithms or the inversion required to form the three-dimensional reconstructions. Alginate beads (AlBes) loaded with labeled mammalian or bacterial cells provide a method of creating defined structures of controllable size and photophysical characteristics and are well-suited for both in vitro and in vivo use. Here we describe a simple and rapid method for efficient and reproducible production of AlBes with specific characteristics and show three example applications with multispectral OA tomography imaging. We show the advantage of AlBes for studying and eventually improving photo-switching OA imaging approaches. As highly defined, homogeneous, quasi point-like signal sources, AlBes might hold similar advantages for studying other agents, light-fluence models, or the impact of detection geometries on correct image formation in the near future.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Chemotherapeutic effects on breast tumor hemodynamics revealed by eigenspectra multispectral optoacoustic tomography (eMSOT).

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice.

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ's function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

Resolution of Spatial and Temporal Heterogeneity in Bevacizumab-Treated Breast Tumors by Eigenspectra Multispectral Optoacoustic Tomography.

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today. SIGNIFICANCE: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.

Multi-Parametric Standardization of Fluorescence Imaging Systems Based on a Composite Phantom.

OBJECTIVE: Fluorescence molecular imaging (FMI) has emerged as a promising tool for surgical guidance in oncology, with one of the few remaining challenges being the ability to offer quality control and data referencing. This paper investigates the use of a novel composite phantom to correct and benchmark FMI systems. METHODS: This paper extends on previous work by describing a phantom design that can provide a more complete assessment of FMI systems through quantification of dynamic range and determination of spatial illumination patterns for both reflectance and fluorescence imaging. Various performance metrics are combined into a robust and descriptive "system benchmarking score," enabling not only the comprehensive comparison of different systems, but also for the first time, correction of the acquired data. RESULTS: We show that systems developed for targeted fluorescence imaging can achieve benchmarking scores of up to 70%, while clinically available systems optimized for indocyanine green are limited to 50%, mostly due to greater leakage of ambient and excitation illumination and lower resolution. The image uniformity can also be approximated and employed for image flat-fielding, an important milestone toward data referencing. In addition, we demonstrate composite phantom use in assessing the performance of a surgical microscope and of a raster-scan imaging system. CONCLUSION: Our results suggest that the new phantom has the potential to support high-fidelity FMI through benchmarking and image correction. SIGNIFICANCE: Standardization of the FMI is a necessary process for establishing good imaging practices in clinical environments and for enabling high-fidelity imaging across patients and multi-center imaging studies.

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Homogentisic acid-derived pigment as a biocompatible label for optoacoustic imaging of macrophages.

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.

Fluorescence imaging reversion using spatially variant deconvolution.

Fluorescence imaging opens new possibilities for intraoperative guidance and early cancer detection, in particular when using agents that target specific disease features. Nevertheless, photon scattering in tissue degrades image quality and leads to ambiguity in fluorescence image interpretation and challenges clinical translation. We introduce the concept of capturing the spatially-dependent impulse response of an image and investigate Spatially Adaptive Impulse Response Correction (SAIRC), a method that is proposed for improving the accuracy and sensitivity achieved. Unlike classical methods that presume a homogeneous spatial distribution of optical properties in tissue, SAIRC explicitly measures the optical heterogeneity in tissues. This information allows, for the first time, the application of spatially-dependent deconvolution to correct the fluorescence images captured in relation to their modification by photon scatter. Using experimental measurements from phantoms and animals, we investigate the improvement in resolution and quantification over non-corrected images. We discuss how the proposed method is essential for maximizing the performance of fluorescence molecular imaging in the clinic.