Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

A case of membranous nephropathy with microspherular deposits progressing to collapsing nephropathy and dialysis dependency, but with full recovery following immunosuppression.

Membranous nephropathy with microspherular deposits is a rare renal condition associated with sub-nephrotic or nephrotic-range proteinuria. We report a case presenting with severe nephrotic syndrome and pathological features of collapsing glomerulopathy. This is the first case we are aware of that progressed to requiring dialysis. The patient received rituximab and corticosteroids. She has now been off dialysis for over a year with both serum creatinine and urine protein-creatinine ratio returning to baseline.

Utility of interval kidney biopsy in ANCA-associated vasculitis.

OBJECTIVES: ANCA-associated vasculitis (AAV) is a rare autoimmune disorder that commonly involves the kidney. Early identification of kidney involvement, assessing treatment-response and predicting outcome are important clinical challenges. Here, we assessed the potential utility of interval kidney biopsy in AAV. METHODS: In a tertiary referral centre with a dedicated vasculitis service, we identified patients with AAV who had undergone interval kidney biopsy, defined as a repeat kidney biopsy (following an initial biopsy showing active AAV) undertaken to determine the histological response in the kidney following induction immunosuppression. We analysed biochemical, histological and outcome data, including times to kidney failure and death for all patients. RESULTS: We identified 57 patients with AAV who underwent at least one interval kidney biopsy (59 interval biopsies in total; median time to interval biopsy ∼130 days). Of the 59 interval biopsies performed, 24 (41%) patients had clinically suspected active disease at time of biopsy which was confirmed histologically in only 42% of cases; 35 (59%) patients were in clinical disease-remission, and this was correct in 97% of cases. The clinician's impression was incorrect in one in four patients. Hematuria at interval biopsy did not correlate with histological activity. Interval biopsy showed fewer acute lesions and more chronic damage compared with initial biopsy and led to immunosuppressive treatment-change in 75% (44/59) of patients. Clinical risk prediction tools tended to operate better using interval biopsy data. CONCLUSION: Interval kidney biopsy is useful for determining treatment-response and subsequent disease management in AAV. It may provide better prognostic information than initial kidney biopsy and should be considered for inclusion into future clinical trials and treatment protocols for patients with AAV.

Tissue-Specific Immunopathology in Fatal COVID-19.

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy.

Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+ transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.

Detection of viral hepatitis E in clinical liver biopsies.

AIMS: To determine the relative utility of in-situ testing for hepatitis E virus (HEV) RNA and paraffin-section polymerase chain reaction (PCR) to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinicopathological characteristics. METHODS AND RESULTS: We evaluated in-situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centres, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histological findings. Thirty-six biopsies from 29 patients had histological data, 27 and 23 of which had satisfactory material for in-situ RNA testing and tissue qPCR, respectively. Both approaches specifically identified HEV infection, but tissue qPCR was significantly more sensitive than RNAscope in-situ testing (P = 0.035). In immunocompetent but not immunosuppressed patients the tissue qPCR yield correlated with the severity of lobular hepatitis (rho = 0.94, P < 0.001). qPCR viral yield was comparably high in allografts and immunosuppressed native livers and significantly greater than with native liver infection. Immunosuppressed patients showed reduced severity of hepatitis and cholestatic changes, compared with immunocompetent patients. Indeed, HEV-infected liver allografts could show minimal hepatitis for many months. In individual cases each technique was useful when serum was not available to identify chronic infection retrospectively (in biopsies taken 4-31 months before diagnosis), to identify persistent/residual infection when contemporary serum PCR was negative and to identify cleared infection. CONCLUSIONS: qPCR is more effective than in-situ RNA testing to identify HEV infection in paraffin-embedded liver biopsies and has diagnostic utility in selected settings.

Benefits of an expanded use of plasma exchange for anti-neutrophil cytoplasmic antibody-associated vasculitis within a dedicated clinical service.

BACKGROUND: Current recommendations for ANCA-associated vasculitis (AAV) support its management within a dedicated clinical service. Therapies for AAV are imperfect with many patients failing to achieve disease control and others experiencing disease relapse. Plasma exchange (PEX) may be beneficial especially when the kidney is involved. METHODS: Within a new, dedicated service we retrospectively assessed, over a 6-year period, the benefits of PEX in two patient cohorts, discriminated by PEX treatment alone. Patients received PEX alongside standard of care if they fulfilled any of the following criteria: 1. serum creatinine >500 µmol/l or dialysis-requiring renal failure, 2. alveolar haemorrhage, 3. renal biopsy showing ≥30 % focal and necrotising lesions ± cellular crescents. Outcome measures included disease remission and relapse, cumulative immunosuppression, and morbidity and mortality. RESULTS: Of 104 new patients, 58 patients received PEX at presentation, 46 did not. Cyclophosphamide and/or rituximab dosing was similar for both groups. Although patients receiving PEX had poorer renal function, a higher C-reactive protein and disease activity score at presentation disease remission rate was similar in both groups (no PEX vs. PEX: 96 % vs. 98 %). The PEX group entered remission quicker (no PEX vs. PEX: 3.9 ± 4.0 vs. 2.8 ± 1.3 months, p < 0.05), with a lower 3-month cumulative glucocorticoid dose (no PEX vs. PEX: 2.5 ± 0.4 vs. 2.3 ± 0.2 g, p < 0.001). Relapse was similar between groups but adverse events lower in the PEX group. CONCLUSIONS: PEX may be of benefit in AAV. Larger, longer randomised controlled trials are now needed.

Radiation dose during coronary angiogram: relation to body mass index.

OBJECTIVE: Ionising radiation based diagnostic and therapeutic cardiology and radiology procedures are very common in present day medical practice and are one of the largest medical sources of radiation to humans. The risk to health from radiation has been extensively documented. Obesity is becoming epidemic not only in the western world, but also in developing countries. In the present study we investigated if a patient's Body Mass Index (BMI) has an effect on the radiation dose received by the patient and operator during diagnostic coronary angiography (CAG). METHODS: We analysed data of 3678 consecutive patients who underwent CAG from September 2007 to April 2010 in our cardiac catheter laboratory. Trans-radial access was used in 622 patients, whereas 3056 patients underwent CAG through trans-femoral route. We calculated the radiation dose in dose area product (DAP) units and correlated it with body mass index, screening time, procedure time, contrast volume, vascular access route and individual operator. RESULTS: Among the explored parameters, body mass index had the most significant association with the radiation dose during the procedure. Despite having similar procedure times and contrast doses, patients with increased BMI received much higher radiation dose during CAG. We also found the left anterior oblique (LAO) caudal and LAO cranial views produced the biggest increase in radiation dose in patients with a high BMI. There was no inter-operator variability. CONCLUSION: Obese patients require more than double the radiation dose in comparison to those with normal BMI. The operator should be aware of the increased dose of radiation required when performing CAG in patients with increased BMI, and especially in LAO cranial and caudal views.

Utility of renal biopsy in the clinical management of renal disease.

Characterizing chronic kidney disease (CKD) at all stages is an essential part of rational management and the renal biopsy plays a key role in defining the processes involved. There remain no global guidelines available to the renal community on indications for this important diagnostic, prognostic, and relatively safe test. Although most nephrologists recognize several clear indications for a renal biopsy, it is still underutilized. It not only helps the clinician to manage the patient with CKD, but it can also help clarify the epidemiology of CKD, and aid research into the pathobiology of disease with the aim of discovering new therapies. It may be useful for instance in elderly patients with CKD, those with diabetes and presumed 'hypertensive nephropathy', and in some patients with advanced CKD as part of the pretransplant work-up. In some populations (for example, immunoglobulin A nephropathy and ANCA vasculitis), renal biopsy allows disease classification that may predict CKD progression and response to therapy. For the individual, interval renal biopsy may be of use in providing ongoing therapeutic and prognostic information. Molecular advances will change the landscape of renal pathology and add a new dimension to the diagnostic precision of kidney biopsy. Organizing the multiplicity of information available in a renal biopsy to maximize benefits to the patient, as well as to the epidemiologist and researcher, is one of the challenges that face the nephrology community.

Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.

Acute antibody-mediated rejection (AMR) occurs in a small minority of sensitized liver transplant recipients. Although histopathological characteristics have been described, specific features that could be used (1) to make a generalizable scoring system and (2) to trigger a more in-depth analysis are needed to screen for this rare but important finding. Toward this goal, we created training and validation cohorts of putative acute AMR and control cases from 3 high-volume liver transplant programs; these cases were evaluated blindly by 4 independent transplant pathologists. Evaluations of hematoxylin and eosin (H&E) sections were performed alone without knowledge of either serum donor-specific human leukocyte antigen alloantibody (DSA) results or complement component 4d (C4d) stains. Routine histopathological features that strongly correlated with severe acute AMR included portal eosinophilia, portal vein endothelial cell hypertrophy, eosinophilic central venulitis, central venulitis severity, and cholestasis. Acute AMR inversely correlated with lymphocytic venulitis and lymphocytic portal inflammation. These and other characteristics were incorporated into models created from the training cohort alone. The final acute antibody-mediated rejection score (aAMR score)--the sum of portal vein endothelial cell hypertrophy, portal eosinophilia, and eosinophilic venulitis divided by the sum of lymphocytic portal inflammation and lymphocytic venulitis--exhibited a strong correlation with severe acute AMR in the training cohort [odds ratio (OR) = 2.86, P < 0.001] and the validation cohort (OR = 2.49, P < 0.001). SPSS tree classification was used to select 2 cutoffs: one that optimized specificity at a score > 1.75 (sensitivity = 34%, specificity = 86%) and another that optimized sensitivity at a score > 1.0 (sensitivity = 81%, specificity = 71%). In conclusion, the routine histopathological features of the aAMR score can be used to screen patients for acute AMR via routine H&E staining of indication liver transplant biopsy samples; however, a definitive diagnosis requires substantiation by DSA testing, diffuse C4d staining, and the exclusion of other insults.

Improved access to histopathology using a digital system could increase the organ donor pool and improve allocation.

Improvements in digital slide scanners have reached a stage that digital whole slide images (WSIs) can be used for diagnostic purposes. A digital system for histopathology, analogous to the systems used in radiology, would allow the establishment of networks of subspecialist histopathologists to provide a regional, national or even international rota to support out of hours histopathology for emergency frozen sections, urgent paraffin sections and to generally improve efficiencies with the provision of histopathology services. Such a system would promote appropriate organ utilization by allowing rapid characterization of unexpected lesions in the donor to determine whether donation should occur and further characterization of the organ, such as the degree of fibrosis in the kidney or steatosis in the liver, to determine whether the organ should be used. If introduced across Europe, this would promote safe and effective exchange of organs and support a cost efficient use of pathologist expertise. This review article outlines current issues with the provision of an urgent out of hours histopathology service and focuses on how such a service has the potential to increase organ donors, improve allocation, sharing and the use of available donor organs.

The value of immunophenotyping hepatocellular adenomas: consecutive resections at one UK centre.

AIMS: To determine the utility of immunophenotyping for classification of hepatocellular adenomas resected at one Scottish centre. METHODS AND RESULTS: This study comprised a retrospective review and immunophenotyping of consecutive resected benign hepatocellular tumours. Fifty-five patients (seven men) had 64 adenomas and 26 focal nodular hyperplasias (FNHs) resected. Map-like glutamine synthetase (GS) staining was specific for FNH. Immunophenotyping changed the morphological typing for three adenomas and resolved 16 of 18 unclassified or equivocal cases, revealing GS positivity in these (seven) and four others. Steatotic/liver fatty acid binding protein-deficient adenomas were the commonest type in women (12/29 women, 41%) but were absent from men. Where one of multiple adenomas was morphologically unclassified, there was still a shared immunophenotype. Diffuse CD34 positivity correlated with GS positivity or unclassified status (P < 0.0001). Supervised cluster analysis identified morphological discriminants for FNH and predictors of adenoma type and their insensitivity in predicting GS status. Forty per cent of men and 7% of women with adenomas had a specific adenoma risk, including danazol and portal venopathies. Inflammatory adenomas were associated with metabolic syndrome, steatosis, or alcohol (P = 0.053). Four patients showed carcinoma ex-adenoma. CONCLUSIONS: The distribution of adenoma types in this population matches that in others, and immunoprofiling is required for accurate typing. Carcinoma ex-adenoma is uncommon and fits the published risk profile (large size and GS-positive).

Hyperglycemia and renin-dependent hypertension synergize to model diabetic nephropathy.

Rodent models exhibit only the earliest features of human diabetic nephropathy, which limits our ability to investigate new therapies. Hypertension is a prerequisite for advanced diabetic nephropathy in humans, so its rarity in typical rodent models may partly explain their resistance to nephropathy. Here, we used the Cyp1a1mRen2 rat, in which the murine renin-2 gene is incorporated under the Cytochrome P4501a1 promoter. In this transgenic strain, administration of low-dose dietary indole-3-carbinol induces moderate hypertension. In the absence of hypertension, streptozotocin-induced diabetes resulted in a 14-fold increase in albuminuria but only mild changes in histology and gene expression despite 28 weeks of marked hyperglycemia. In the presence of induced hypertension, hyperglycemia resulted in a 500-fold increase in albuminuria, marked glomerulosclerosis and tubulointerstitial fibrosis, and induction of many of the same pathways that are upregulated in the tubulointerstitium in human diabetic nephropathy. In conclusion, although induction of diabetes alone in rodents has limited utility to model human diabetic nephropathy, renin-dependent hypertension and hyperglycemia synergize to recapitulate many of the clinical, histological, and gene expression changes observed in humans.

Survey of liver pathologists to assess attitudes towards digital pathology and artificial intelligence.

AIMS: A survey of members of the UK Liver Pathology Group (UKLPG) was conducted, comprising consultant histopathologists from across the UK who report liver specimens and participate in the UK National Liver Pathology External Quality Assurance scheme. The aim of this study was to understand attitudes and priorities of liver pathologists towards digital pathology and artificial intelligence (AI). METHODS: The survey was distributed to all full consultant members of the UKLPG via email. This comprised 50 questions, with 48 multiple choice questions and 2 free-text questions at the end, covering a range of topics and concepts pertaining to the use of digital pathology and AI in liver disease. RESULTS: Forty-two consultant histopathologists completed the survey, representing 36% of fully registered members of the UKLPG (42/116). Questions examining digital pathology showed respondents agreed with the utility of digital pathology for primary diagnosis 83% (34/41), second opinions 90% (37/41), research 85% (35/41) and training and education 95% (39/41). Fatty liver diseases were an area of demand for AI tools with 80% in agreement (33/41), followed by neoplastic liver diseases with 59% in agreement (24/41). Participants were concerned about AI development without pathologist involvement 73% (30/41), however, 63% (26/41) disagreed when asked whether AI would replace pathologists. CONCLUSIONS: This study outlines current interest, priorities for research and concerns around digital pathology and AI for liver pathologists. The majority of UK liver pathologists are in favour of the application of digital pathology and AI in clinical practice, research and education.

The use of digital magnification to reduce radiation dose in the cardiac catheter laboratory.

OBJECTIVES: To audit whether using magnification of images by use of a large viewing screen using digital matrix magnification which enlarges the image by 33% without using the X-ray machine zoom magnification protocols on a Siemens Artis Zee X-ray machine in a cardiac catheter laboratory results in a reduction of kerma-area product (KAP) for both diagnostic and interventional procedures. This reduction was predicted in an in vitro study in our laboratory, which has previously shown a 20.4% reduction in KAP. METHODS: A retrospective analysis was conducted of the radiation exposure to compare the measured KAP recorded during the period when conventional magnification with automatic brightness and dose control was used on a Siemens Artis Zee X-ray machine with a flat panel detector and when magnification settings were avoided by using a large screen to enlarge and project a non-magnified image by digital magnification. The analysis was carried out for patients having a diagnostic coronary angiogram and those having an interventional coronary procedure. RESULTS: For diagnostic coronary angiograms the median KAP per procedure in the period using conventional magnification was 2124.5 µGy.m2 compared to 1401 µGy.m2 when image matrix magnification was used, a 34% reduction (p < 0.0001). For interventional coronary procedures, the median KAP per procedure in the period using conventional magnification was 3791 µGy.m2 compared to 2568.5 µGy.m2 when image matrix magnification was used, a 32% reduction (p < 0.0001). CONCLUSION: Avoiding using conventional magnification in the cardiac catheter laboratory and using a large screen to magnify images was associated with a statistically significant greater than 30% reduction in KAP. ADVANCES IN KNOWLEDGE: This paper is the proof in clinical practice of a theoretical conclusion that radiation dose (KAP) is reduced by use of Image matrix magnification using a large viewing screen without the need to use X-ray tube magnification without significant loss of image resolution in interventional cardiology. The same approach will be useful in interventional radiology.

Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease.

In a recent phase II clinical trial using banked allogeneic CTL lines to treat EBV-associated posttransplant lymphoproliferative disease, a response rate of 52% was recorded 6 mo posttreatment. Tumor response was associated with an increase in both CTL/recipient HLA matches and CD4(+) T cells within the infused CTL lines. The present study was undertaken to correlate tumor response with CTL specificity. The majority of CTL lines infused recognized EBV-encoded nuclear Ag-3 proteins, but CTL protein specificity itself did not correlate with tumor response. Specificity in conjunction with donor/recipient functional HLA matching as opposed to HLA matching alone, however, was important for tumor response. CTL receptor TCR beta-chain variable gene subfamilies were polyclonal, with no preferential use of a particular family. However, tumor response was improved in those receiving CTL lines with polyclonal vs clonal distribution for subfamilies 2, 3, and 9. Interestingly, in five of six tumors (five Hodgkin's-like and one Burkitt's-like posttransplant lymphoproliferative disease) with restricted viral gene expression a complete response was recorded, although in some cases the tumor cells did not express the proteins recognized by the infused CTL. Thus CTL were advantageous when functionally HLA matched but for certain tumor types complete responses occurred in the absence of detectable specific CTL/tumor recognition. We suggest that either the allogenic CTL contained small, undetectable, EBV-specific, HLA-matched T cell populations or perhaps they stimulated nonspecific inflammatory responses in vivo, which were beneficial for tumor regression. These observations should be considered when designing and implementing CTL therapies.

Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver.

BACKGROUND: Stem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling. OBJECTIVE: To examine the composition and formation of stem progenitor cell niches. METHODS: The composition of the hepatic progenitor cell niche in independent models of liver injury and hepatic progenitor cell activation in rodents and humans was studied. To identify the origin of the progenitor and niche cells, sex-mismatched bone marrow transplants in mice, who had received the choline-ethionine-deficient-diet to induce liver injury and progenitor cell activation, were used. The matrix surrounding the progenitor cells was described by immunohistochemical staining and its functional role controlling progenitor cell behaviour was studied in cell culture experiments using different matrix layers. RESULTS: The progenitor cell response in liver injury is intimately surrounded by myofibroblasts and macrophages, and to a lesser extent by endothelial cells. Hepatic progenitor cells are not of bone marrow origin; however, bone marrow-derived cells associate intimately with these cells and are macrophages. Laminin always surrounds the progenitor cells. In vitro studies showed that laminin aids maintenance of progenitor and biliary cell phenotype and promotes their gene expression (Dlk1, Aquaporin 1, gammaGT) while inhibiting hepatocyte differentiation and gene expression (CEPB/alpha). CONCLUSIONS: During liver damage in rodents and humans a stereotypical cellular and laminin niche forms around hepatic progenitor cells. Laminin helps maintenance of undifferentiated progenitor cells. The niche links the intrahepatic progenitor cells with bone marrow-derived cells and links tissue damage with progenitor cell-mediated tissue repair.

Tissue-resident macrophages protect the liver from ischemia reperfusion injury via a heme oxygenase-1-dependent mechanism.

Kupffer cells are the resident macrophage population of the liver and have previously been implicated in the pathogenesis of hepatic ischemia-reperfusion injury (IRI). Kupffer cells are the major site of expression of hepatic heme oxygenase-1 (HO-1), which has been shown to have anti-inflammatory actions and to protect animals and cells from oxidative injury. Kupffer cells and circulating monocytes were selectively ablated using liposomal clodronate (LC) in the CD11b DTR mouse before induction of hepatic ischemia. Kupffer cell depletion resulted in loss of HO-1 expression and increased susceptibility to hepatic IRI, whereas ablation of circulating monocytes did not affect IRI phenotype. Targeted deletion of HO-1 rendered mice highly susceptible to hepatic IRI. In vivo, HO-1 deletion resulted in pro-inflammatory Kupffer cell differentiation characterized by enhanced Ly6c and MARCO (macrophage receptor with collagenous structure) expression as well as decreased F4/80 expression, mirrored by an expansion in immature circulating monocytes. In vitro, HO-1 inhibition throughout macrophage differentiation led to increased cell numbers, and pro-inflammatory Ly6c+ CD11c- F4/80- phenotype. These data support a critical role for tissue-resident macrophages in homeostasis following ischemic injury, and a co-dependence of HO-1 expression and tissue-resident macrophage differentiation.