Background: A subgroup of COPD patients (pts) displays eosinophilia, a trait targeted in several clinical drug programs. Little is known about the mechanisms underlying eosinophil (eos) infiltration into COPD lungs, or how eos infiltration relates to microbial presence and immune cell composition. Aims: Reveal the spatial relationship between tissue eos, microbes, and immune cell patterns in lungs and lymph nodes from COPD pts. Methods: Surgical lung tissue was collected from 35 COPD pts (GOLD I-IV) and 17 non-COPD controls. Microbes and complete immune cell profiles were identified histologically by combined in situ hybridization and multiplex immunohistochemistry, followed by computerized spatial analysis. Results: Mean total tissue eos increased in severe COPD. The infiltration pattern was patchy with no spatial correlation to bacteria, viruses, or fungi. Multiplex imaging of major leukocyte and structural cell populations revealed spatially distinct immune cell niches in which eos-rich microenvironments occurred alongside neutrophil- and macrophage-dominated regions. Eos and type 2 foci were spatially linked to basophils, CD20+ B lymphocytes, CD20-CD19+ plasmablasts, and CD138+ plasma cells. Eos infiltration in bronchi, small airways, or alveolar regions was associated with eosinophilia in the subcapsular and medullary lymphatic sinuses in lung-draining lymph nodes. Conclusions: Patchy eosinophilia was linked to adaptive immune niches that with spatially separated non-eos inflammation yields complex mixed inflammatory signatures that likely impact treatment response. The finding of no spatial association between microbial presence and eosinophilia suggests that other non-infectious factors are involved.

Eosinophils are commonly associated with Th2-type inflammation as occurs in allergic diseases such as asthma. However, eosinophils are, also, present in vivo in Th1-type environments found in translational models of infection, cancer, or transplant. Little is known regarding type 2 and type 1 immune phenotypes of mouse and human eosinophils. The goal was to compare mouse and human eosinophil immune phenotypes following exposure to Th2 and Th1 cytokines.

Eosinophils have specific immune phenotypes in type 2 and type 1 environments both in vitro and in translational models of disease in vivo. The regulatory transcription factors (TFs) that control eosinophil activation into type 2 or type 1 immune phenotypes (E2 or E1, respectively) are unknown. The goal was to utilize transcriptome data to identify candidate TFs and test their role in eosinophil immune phenotypes. RNA sequencing was completed on wild type mouse and healthy human peripheral blood eosinophils cultured with cytokines that represent a Th2 or Th1-associated environment. Sorted lung eosinophils from rejecting lung allografts, which is a TNFα/IFNγ rich environment, underwent RNA sequencing. Upregulated genes were analyzed with ChEA3 TF enrichment analysis to identify the top 20 TFs. Interferon regulator factor 1 (IRF1)-deficient eosinophils were cultured with Th2 or Th1 cytokines. Of the top 20 TFs, E2 and E1 eosinophils had five and six unique TFs, respectively. Eosinophils from rejecting lung allografts shared five of the six E1-unique TFs, one of which was IRF1. Generation of bone marrow-derived IRF1-knockout eosinophils resulted in similar expansion and differentiation as compared to wild type eosinophils. Counterintuitively, culture of IRF1-deficient eosinophils with Th1 cytokines led to significant type 2 cytokine and chemokine production (e.g., IL-4, IL-13, and CCL17) and cell surface expression that would otherwise not occur in wild type eosinophils exposed to TNFα/IFNγ. Mouse and human eosinophils share several regulatory TF pathways. IRF1 is critical in regulating type 2 and type 1 immune responses of eosinophils in Th1 environments.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1–2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in a population of genetically identical cells, and performed protein expression correlation analysis to identify co-regulated proteins. We also profiled >6,000 neurons in a human formalin-fixed paraffin-embedded (FFPE) hippocampus tissue. By partitioning these neurons into varied cell clusters based on their multiplexed protein expression profiles, we observed different sub-regions of the hippocampus consist of neurons from distinct clusters.

Innate type 2 lymphoid cells (ILC2s) recruit and accumulate in the lung in response to type 2 inflammation. This coincides with production of IL-5 and IL-13 from ILC2s and pulmonary eosinophilia. Although ILC2s have been shown to modulate eosinophil activities, the role of eosinophils in regulating ILC2 responses is less well defined and may represent a novel regulatory feedback pathway. Type 2 pulmonary inflammation was induced by either intratracheal cytokine administration (e.g., IL-33) or using models of ovalbumin or house dust mite allergen sensitization/challenge. Eosinophils were specifically depleted immediately prior to instillation or challenge using inducible eosinophil-deficient mice (iPHIL) mice to determine the role of eosinophils on pulmonary ILC2s in these type 2 inflammation models. Lung-derived ILC2s were cultured with eosinophils to define ILC2 and eosinophil interactions. Activation and chemotaxis of ILC2s were assessed in vitro. Depletion of eosinophils in all type 2 models of respiratory inflammation resulted in a significant reduction of total and activated pulmonary ILC2s. For example, lung IL-13+ILC2s were significantly reduced in IL-33 treated eosinophil-depleted iPHIL mice (236,018± 42,307 vs 106,220 ±26,617 IL-13+ILC2s (p<0.05)). Baseline and saline treated animals had comparable pulmonary ILC2 numbers (<20,000 ILC2s (p>0.05) between eosinophil-proficient and deficient mice. In vitro IL-33 activated eosinophils released chemotactic factors for ILC2s and induced activation of ILC2s through cell-cell contact. Our data demonstrates an underappreciated and significant role for eosinophils in recruitment and activation of ILC2s. These data suggest a reciprocal role for eosinophil-ILC2 interactions in amplification of the type 2 pulmonary inflammatory response.

Under homeostatic conditions and in disease progression eosinophils have a variety of effector functions regulating Local Immunity And/or Remodeling/Repair (LIAR hypothesis). These activities are manifestations of different eosinophil subtypes, which, in turn, depend on the cytokine milieu of the tissue microenvironment. Eosinophil subtypes are characterized by expression of specific genes and we hypothesize that their functions are supported by a distinct metabolic phenotype. The goal of this study was to examine relationships between Th1 or Th2 cytokine-dependent gene expression and parameters of energy metabolism in mouse eosinophils. Eosinophils from IL-5 overexpressing transgenic mice (NJ1638) were purified to >98% purity and cultured in cytokine cocktails typical for Th1 or Th2 environment to polarize the cells to their subtypes (E1 and E2). RNA was extracted for RNA-seq transcriptome analysis and mitochondrial function was assessed by measuring the relative uptake of MitoTracker Orange (potential dependent) to MitoTracker Far Red (potential independent). Rates of oxygen consumption and extracellular acidification were directly assessed in polarized eosinophils using a Seahorse XFe96 Analyzer. RNA-seq analysis showed elevated expression of genes present in mitochondrial respiratory chain in Th2 compared to Th1 cytokine treated eosinophils. Consistent with these observations E2 eosinophils had a higher inner mitochondrial membrane potential. E2 eosinophils had significantly higher maximal respiratory capacity and rate of media acidification compared to E1 eosinophils. Our studies showed an overall elevated metabolic rate in Th2 cytokine treated eosinophils (E2 eosinophils), suggesting a link between eosinophil subtypes and parameters of their energy metabolism.

Eosinophils are evolutionarily conserved granulocytes typically associated with parasite killing or allergic diseases. Although eosinophils are traditionally characterized as destructive and cytotoxic cells with the main activity being degranulation (releasing toxic proteins), we and others are identifying eosinophils as immune regulatory cells in health and disease. We hypothesize that there are many subtypes of immune polarized tissue infiltrating eosinophils that are disease/tissue specific and can be used as a diagnostic/prognostic indicator of health and disease. In particular, we propose type 1 and type 2 immune environments induce specific gene expression and functions of eosinophils. Blood-derived eosinophils were purified from IL-5 over expressing mice (NJ.1638) and cultured for 18 hours with type 2 cytokines IL-33/GM-CSF/IL-4 or type 1 cytokines IFNγ/TNFα to generate E2 and E1 eosinophils respectively. RNAseq was completed with confirmation RT-PCR. Cell surface markers were assayed by flow cytometry. Viability was compared with and without corticosteroids. Proteins were assayed by multiplex assay and degranulation by eosinophil peroxidase (EPX) ELISA. RNAseq analysis showed E2 as compared to E1 had 371 upregulated and 407 downregulated genes and E1 as compared to E2 had 386 upregulated and 107 downregulated genes. Released proteins were unique between subtypes. Characteristic cell surface markers of E2 include Cd11bhi and CD69+ while E1 include Ly6Chi. E1 eosinophils had reduced viability with and without corticosteroid treatment as compared to E2 eosinophils. E2 eosinophils released significantly more EPX upon culture. The cytokine environment induces differential activation of eosinophils resulting in unique gene expression and functional activities.

The ability to profile transcripts and genomic loci comprehensively in single cells in situ is essential to advance our understanding of normal physiology and disease pathogenesis. Here we report a highly multiplexed single-cell in situ RNA and DNA analysis approach using bioorthogonal cleavable fluorescent oligonucleotides. In this approach, oligonucleotides tethered to fluorophores through an azide-based cleavable linker are used to detect their nucleic acids targets by in situ hybridization. After fluorescence imaging, the fluorophores in the whole specimen are efficiently cleaved in 30 minutes without loss of RNA or DNA integrity. Through reiterative cycles of hybridization, imaging, and cleavage, this method has the potential to quantify hundreds to thousands of different RNA species or genomic loci in single cells in situ at the single-molecule sensitivity. Applying this approach, we demonstrate that different nucleic acids can be detected in each hybridization cycle by multi-color staining, and at least ten continuous hybridization cycles can be carried out in the same specimen. We also show that the integrated single-cell in situ analysis of DNA, RNA and protein can be achieved using cleavable fluorescent oligonucleotides combined with cleavable fluorescent antibodies. This highly multiplexed imaging platform will have wide applications in systems biology and biomedical research.

Bioorthogonal cleavable fluorescent oligonucleotides have been developed to enable highly multiplexed single-cell in situ RNA and DNA analysis.