Blood immune cell profiling in adults with longstanding type 1 diabetes is associated with macrovascular complications.

Aims/hypothesis: There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity. Methods: Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects. Results: Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications. Conclusions: Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications.

Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3-6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

Immune phenotyping in a pediatric multicenter transplant study: Suitability of a preformulated dry-antibody panel system.

Flow-cytometric immune phenotyping is influenced by cryopreservation and inter-laboratory variability limiting comparability in multicenter studies. We assessed a system of optimized, pre-mixed dry-antibody panel tubes requiring small amounts of whole blood for validity, reliability and challenges in a Canadian multicenter study (POSITIVE) with long-distance sample shipping, using standardized protocols. Thirty-seven children awaiting solid-organ transplant were enrolled for parallel immune-phenotyping with both validated, optimized in-house panels and the dry-antibody system. Samples were collected before, 3 and 12 months post-transplant. Quality-assurance measures and congruence of phenotypes were compared using Bland-Altman comparisons, linear regression and group comparisons. Samples showed excellent lymphocyte viability (mean 94.8 %) and recovery when processed within 30 h. Comparing staining methods, significant correlations (Spearman correlation coefficient >0.6, p < 0.05), mean difference <5 % and variation 2SD <25 % were found for natural-killer, T and B cells, including many immunologically important cell subsets (CD8+, naïve, memory CD4+ T; switched-memory, transitional B). Some subgroups (plasmablasts, CD1d+CD5hi B cells) showed weak correlations, limiting interpretation reliability. The dry-antibody system provides a reliable method for standardized analysis of many immune phenotypes after long-distance shipping when processed within 30 h, rendering the system attractive for pediatric studies due to small blood amounts required and highly standardized processing and analysis.

Isolation and identification of primary cells: A comprehensive primary cell culture experiment for graduate students.

Experimental teaching is an important part of postgraduate training in basic and clinical medicine. While primary cell isolation and identification are among the most important research techniques for medical graduate students, most graduate students do not understand and master these techniques before starting their research experience. In particular, many students lack training in this field, and high-quality teaching and learning materials are still very sparse. Here, we designed a practical experiment course for graduate students engaged in research. The target students usually have research projects involving primary cell culture in their future research, making the course highly applicable for the students. The lab exercise focused on the methods of primary cell isolation (including mechanical grinding method, explant culture method and enzymatic digestion method) and identification (including flow cytometry, immunofluorescence, and periodic acid-Schiff (PAS) staining). It aimed to help students master the conceptual, principle, technical, operation, and analytical skills related to primary cell culture and contributed to their foundation for future research. Students generally reflect that they have initially mastered the isolation and identification of primary cell culture as a result of the course. Student feedback also indicates significantly increased confidence in the practical application of primary cell culture in the future. Here, we provide our experience for others who may want to implement similar courses.

Adipocyte ABCA1 expression analysis using flow cytometry.

Adipocyte characterization and assessing membrane proteins using flow cytometry has been proven to be challenging as adipocytes are fragile, especially in subjects with high BMI. We overcame these challenges through a protocol optimizing tissue digestion time by reducing intermediate steps to minimize adipocyte friction and breakage. We avoided requirement for specialized instrument configuration and used a modified gating strategy to prevent inclusion of lipid droplets during analysis. Up to 90% of the cell population were available in the gating area. We checked the expression level of ABCA1, a membrane protein reaffirming adipocyte selection. In summary, this protocol requires lesser tissue sample improving feasibility and cost efficiency. Thus, our flow cytometry method is an improvement for studying adipocyte membrane characteristics.

Flow Cytometric Identification of Human IgE+ B Lineage Subsets.

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested.

Intracellular Flow Cytometry ("Phosphoflow") to Assess Signal Transduction in Rare Populations Such As Memory B Cell Subsets and Plasma Cells.

Intracellular flow cytometry is a powerful technique that can be used to interrogate signalling in rare cellular populations. The strengths of the technique are that massively parallel readouts can be gained from thousands of single cells simultaneously, and the assay is fast and relatively straightforward. This plate-based protocol enables different doses and different timepoints of stimulation to be assessed and has been optimized for rare B cell populations. Combining this technique with high-dimensional flow cytometry enables multiple signalling proteins to be measured with high confidence.

Indeterminate measurable residual disease by multiparameter flow cytometry is associated with an intermediate risk of clinical relapse in adult patients with acute leukaemia.

Measurable residual disease (MRD) is useful for prognostication and for monitoring response to treatment in patients with acute leukaemia. MRD by multiparametric flow cytometry (MFC-MRD) utilises the leukaemia-associated immunophenotype (LAIP) and difference from normal (DfN) strategies to identify the leukaemic clone. Difficulties arise when the LAIP overlaps with normal regeneration, there is clonal evolution, or when the abnormal clone population is exceptionally small e.g., <0.01% of CD45+ cells. Such cases are reported as 'indeterminate'; however, there is little international consensus on this reporting. The relationship between clinical outcomes and indeterminate MFC-MRD is unknown. Here we determine the rate of indeterminate MFC-MRD reporting, its relationship to concurrent molecular MRD results when available, and to clinical outcomes to 12 months. We performed an internal audit of all adult testing for MFC-MRD between January and December 2021. A total of 153 consecutive patients with a diagnosis of acute leukaemia were included. Successive MFC-MRD results and clinical outcomes were recorded over a 12-month period from time of inclusion into the study. In total, 460 MFC-MRD tests from 153 patients were reviewed and 73 (16%) MFC-MRD tests from 54 (35%) patients were reported as indeterminate. The majority (70%) were at low levels between 0.01-0.1% of CD45+ cells. Compared to patients with a negative result, acute myeloid leukaemia (AML) was more frequent in patients who had an indeterminate MFC-MRD (70% vs 36%), and B-cell acute lymphoblastic leukaemia was less common (20% vs 55%). In patients with indeterminate MFC-MRD results, one-third had received either chemotherapy or allogeneic haemopoietic stem cell transplant (aHSCT) within the preceding 3 months. Agreement between MFC and molecular MRD testing was low. Patients with indeterminate MFC-MRD had leukaemia relapse rates below patients with a positive MFC-MRD, but greater than those with negative MFC-MRD (positive 33% vs indeterminate 21% vs negative 8%, p = 0.038). Overall, these findings indicate that indeterminate MFC-MRD results are more common in adults with AML and also in those who have received chemotherapy or aHSCT within the previous 3 months. We report for the first time that indeterminate MFC-MRD is a finding of potential clinical significance, which associates with a numerically higher median relapse rate within 12 months when compared to a negative MFC-MRD result.

Clinical and cytological characteristics of serous effusions in 69 cases of lymphoma patients.

BACKGROUND: To explore the value of cell morphology, immunophenotype, and gene alterations of serosal effusion in the diagnosis of lymphoma. METHODS: Serosal effusion of 69 cases of lymphoma patients were collected, including 36 cases with malignant effusion and 33 cases with nonmalignant effusion. Ordinary cytology, liquid-based cytology, cellblock, and immunocytochemical staining were performed in each case, some cases were detected by fluorescence in situ hybridization for C-MYC, BCL2, and BCL6 gene translocations. T/B cell ratio in malignant and nonmalignant serosal effusions was analyzed and compared by flow cytometry (FCM) and immunohistochemical (IHC), respectively. The prognostic value of serous effusion in diffuse large B-cell lymphoma (DLBCL) was investigated and another 20 DLBCL cases without effusion were successively selected as control. RESULTS: The number of naive lymphocytes, apoptotic bodies, and mitotic figures were more common in malignant effusions compared with nonmalignant effusions (p < .01). The top three lymphomas in malignant effusion were DLBCL (19/36, 52.8%), mantle cell lymphoma (MCL) (4/36, 11.1%, 3 blastoid variant) and high-grade B-cell lymphoma (HGBL) (4/36, 11.1%). T/B cell ratio by FCM analysis ranged from 0.00 to 0.55 (mean 0.084) in malignant effusion, and 2.58 to 984.00 (mean 249.9) in nonmalignant effusion. The difference was significant (p = .017). The T/B cell ratio by IHC analysis ranged from 0.02 to 3.00 (mean 0.200) in malignant effusion, and 2.00-100.00 (mean 34.10) in nonmalignant effusion. The difference was significant (p = .017). In the effusions involving DLBCL, most effusions were present at the time of diagnosis (57.9%); single pleural effusions were more common (36.8%). The median overall survival times of patients with malignant effusion, nonmalignant effusion and DLBCL without serous effusion were 11, 17, and 23 months respectively (p = .04). Three patients of HGBL died, and the overall survival times were 5, 8, and 9 months, respectively. CONCLUSIONS: The cytomorphological characteristics combined with immunophenotype, FCM, gene rearrangement, and other tests can diagnose and classify patients with effusion as the first symptom. The T/B cell ratio is less than 1 by FCM or IHC suggesting a malignant serosal effusion. The presence of malignant effusion in DLBCL patients is an important clue for poor prognosis.

Protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts.

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting. For complete details on the use and execution of this protocol, please refer to Zhang et al.,1 Valkov et al.,2 Vang et al.,3 and Li et al.4.

Evaluation of a flow cytometry-based method for determination of T-lymphocyte subtypes for quality assessment of cell therapy products.

Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C (p = .004) and at room temperature (p = .018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.

Eosinophils in hidradenitis suppurativa patients exhibit pro-inflammatory traits, implicating a potential pathogenic role in the disease.

Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by painful nodules, abscesses and purulent secretions in intertriginous regions. Intense pruritus frequently accompanies HS lesions, adding further discomfort for patients. While Th17 pathway activation is implicated in HS pathogenesis, disease mechanisms are still not fully understood, and therapeutics are lacking. Previous reports raise a potential role for eosinophils in HS, showing a strong association of eosinophil levels with disease severity. To investigate eosinophils in HS, we recruited patients and matched healthy controls and then performed flow-cytometry studies, eosinophil stimulation assays, and lesional skin staining for eosinophils. We found that HS patients reported similar levels of pain and itch. Compared to matched controls, HS blood exhibited decreased mature eosinophils and increased numbers of immature eosinophils, coupled with a significant increase in dermal eosinophilic infiltrates. Additionally, IL-17RA+ eosinophils were highly and significantly correlated with multiple HS-related clinical scores. In both stimulated and unstimulated conditions, HS eosinophils showed an inflammatory phenotype versus controls, including an increase in costimulatory T- and B-cell markers (e.g. CD5 and CD40) following all stimulations (TNFα/IL-17A/IL-17F). These findings highlight the significance of pruritus in HS and suggest a higher turnover of eosinophils in HS blood, potentially due to the consumption of eosinophils in skin lesions. Our data delineate the features and functions of eosinophils in HS and suggest that eosinophils participate in disease pathogenesis, advancing Th17-related inflammation. Further studies are needed to investigate eosinophils' response to current HS treatments and their potential as a therapeutic target in the disease.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

GateNet: A novel neural network architecture for automated flow cytometry gating.

BACKGROUND AND OBJECTIVE: Flow cytometry is a widely used technique for identifying cell populations in patient-derived fluids, such as peripheral blood (PB) or cerebrospinal fluid (CSF). Despite its ubiquity in research and clinical practice, the process of gating, i.e., manually identifying cell types, is labor-intensive and error-prone. The objective of this study is to address this challenge by introducing GateNet, a neural network architecture designed for fully end-to-end automated gating without the need for correcting batch effects. METHODS: For this study a unique dataset is used which comprises over 8,000,000 events from N = 127 PB and CSF samples which were manually labeled independently by four experts. Applying cross-validation, the classification performance of GateNet is compared to the human experts performance. Additionally, GateNet is applied to a publicly available dataset to evaluate generalization. The classification performance is measured using the F1 score. RESULTS: GateNet achieves F1 scores ranging from 0.910 to 0.997 demonstrating human-level performance on samples unseen during training. In the publicly available dataset, GateNet confirms its generalization capabilities with an F1 score of 0.936. Importantly, we also show that GateNet only requires ≈10 samples to reach human-level performance. Finally, gating with GateNet only takes 15 microseconds per event utilizing graphics processing units (GPU). CONCLUSIONS: GateNet enables fully end-to-end automated gating in flow cytometry, overcoming the labor-intensive and error-prone nature of manual adjustments. The neural network achieves human-level performance on unseen samples and generalizes well to diverse datasets. Notably, its data efficiency, requiring only ∼10 samples to reach human-level performance, positions GateNet as a widely applicable tool across various domains of flow cytometry.

Unlocking the secrets of Hofbauer cells in placental (patho-) physiology: Isolation and quality assessment in human term placenta.

INTRODUCTION: Hofbauer cells (HBCs) are macrophages of fetal origin that reside in the villous tissue. They are the only immune cells within healthy villi. While HBCs perform innate immune functions such as phagocytosis and antigen presentation, they are increasingly recognized for their diverse roles in placental physiology e.g. vascular functionality, tissue homeostasis, tolerance. Consequently, HBCs are of utmost interest in a variety of non-physiological placental conditions. ISOLATION: Villous tissue is collected freshly after delivery and finely minced. The resulting tissue is digested in a two-step process, using Trypsin/DNase to separate cytotrophoblasts and collagenase/DNase to penetrate deeper into the villous stroma, containing HBCs, and obtain a single cell suspension. After a density gradient centrifugation, the corresponding cell layer is collected and subjected to negative immune selection of HBCs, yielding unaffected cells that have not been activated during the isolation process. QUALITY CONTROL: In addition to a classical immunocytochemistry (ICC) approach including macrophage markers, and markers for potentially contaminating cell types (e.g. fibroblasts, muscle, mesenchymal cells), we have developed a multi-color flow cytometry (FC) panel. This panel assesses Hofbauer cell purity and polarization states more accurately and comprehensively than qualitative ICC, using percentage analysis of parent cells to estimate the expression levels of specific markers. DISCUSSION: The presented protocol allows us to isolate HBCs in significant numbers and high purity, even from placentae compromised by preeclampsia (PE) with limited placental volume. We have successfully developed and implemented this protocol to study healthy, diabetic and PE macrophages, aiding a better understanding of the underlying placental pathophysiology at the cellular level.

A Redox-Shifted Fibroblast Subpopulation Emerges in the Fibrotic Lung.

Idiopathic pulmonary fibrosis (IPF) is an aggressive and thus far incurable disease, characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis, and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population-specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)Hhigh and NAD(P)Hlow sub-populations. NAD(P)Hhigh fibroblasts exhibited elevated pro-fibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)Hlow fibroblasts. The NAD(P)Hhigh population was present in healthy lungs but expanded with time after bleomycin injury suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)Hhigh cells in freshly dissociated lungs of subjects with IPF relative to controls, and similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.