The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study.

Introduction: Chronic rejection is a major complication post-transplantation. Within lung transplantation, chronic rejection was considered as airway centred. Chronic Lung Allograft Dysfunction (CLAD), defined to cover all late chronic complications, makes it more difficult to understand chronic rejection from an immunological perspective. This study investigated the true nature, timing and location of chronic rejection as a whole, within mouse lung transplantation. Methods: 40 mice underwent an orthotopic left lung transplantation, were sacrificed at day 70 and evaluated by histology and in vivo µCT. For timing and location of rejection, extra grafts were sacrificed at day 7, 35, 56 and investigated by ex vivo µCT or single cell RNA (scRNA) profiling. Results: Chronic rejection originated as innate inflammation around small arteries evolving toward adaptive organization with subsequent end-arterial fibrosis and obliterans. Subsequently, venous and pleural infiltration appeared, followed by airway related bronchiolar folding and rarely bronchiolitis obliterans was observed. Ex vivo µCT and scRNA profiling validated the time, location and sequence of events with endothelial destruction and activation as primary onset. Conclusion: Against the current belief, chronic rejection in lung transplantation may start as an arterial response, followed by responses in venules, pleura, and, only in the late stage, bronchioles, as may be seen in some but not all patients with CLAD.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Comparison of the single-cell and single-nucleus hepatic myeloid landscape within decompensated cirrhosis patients.

Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.

The dynamics of cytokine release during 24 hours continuous normothermic machine perfusion liver preservation: An explorative porcine study.

BACKGROUND: Normothermic machine perfusion (NMP) has been proposed to preserve liver grafts in a less pro-inflammatory environment. However, the effect of NMP on liver inflammation remains unclear. Therefore, we aimed at characterizing the inflammatory response during continuous NMP with a comprehensive investigation of cytokine release during perfusion. METHODS: Ten porcine livers underwent either 24 h NMP or whole blood-based NMP (WB-NMP) immediately after procurement. WB-NMP was used as a positive control to mimic early post-reperfusion inflammation. High mobility group box-1 (HMGB1), interleukin 1-beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha), interleukin 6 (IL-6), 8 (IL-8), and 10 (IL-10), transforming growth factor-beta (TGFbeta), aspartate transferase (AST), and hyaluronic acid were measured in the perfusate. The area under the curve (AUC) of their perfusate concentration was compared between groups. Median (IQR) is given. RESULTS: The AUC of HMGB1 and IL-1beta was similar between groups. Compared to WB-NMP, NMP inhibited the release of TNFalpha [NMP: 20275 (18402-32 152), WB-NMP: 242100 (203511-244 238); p = 0.01], IL-6 [NMP: 1206 (338.9-1686), WB-NMP: 8444 (7359-10 087); p = 0.03], and IL-8 [NMP: 1635 (106.90-2130), WB-NMP: 3951 (3090-4116); p = 0.008]. The release of TGFbeta remained unchanged but IL-10 release was lower in NMP [1612 (1313-1916), WB-NMP: 5591 (4312-6421); p = 0.01]. The ratios TGFbeta:TNFalpha and IL-10:TNFalpha were significantly higher in the NMP than in the WB-NMP group. Importantly, the AUC of AST was significantly lower during NMP [1960 (1950-2893)] than WB-NMP [6812 (6370-7916); p = 0.02]. CONCLUSIONS: Continuous NMP leads to the release of detectable levels of cytokines with a slow, linear increase over time and a shift toward anti-inflammatory signaling.

Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.

The Distinct Innate Immune Response of Warm Ischemic Injured Livers during Continuous Normothermic Machine Perfusion.

Although normothermic machine perfusion (NMP) provides superior preservation of liver grafts compared to static cold storage and allows for viability testing of high-risk grafts, its effect on the liver immune compartment remains unclear. We investigated the innate immune response during 6 h of continuous NMP (cNMP) of livers that were directly procured (DP, n = 5) or procured after 60 min warm ischemia (WI, n = 5), followed by 12 h of whole blood (WB) reperfusion. WI livers showed elevated transaminase levels during cNMP but not after WB reperfusion. Perfusate concentrations of TNF-α were lower in WI livers during cNMP and WB reperfusion, whereas IL-8 concentrations did not differ significantly. TGF-ß concentrations were higher in WI livers during NMP but not after WB reperfusion, whereas IL-10 concentrations were similar. Endoplasmic stress and apoptotic signaling were increased in WI livers during cNMP but not after WB reperfusion. Additionally, neutrophil mobilization increased to a significantly lesser extent in WI livers at the end of NMP. In conclusion, WI livers exhibit a distinct innate immune response during cNMP compared to DP livers. The cytokine profile shifted towards an anti-inflammatory phenotype during cNMP and WB reperfusion, and pro-apoptotic signaling was stronger during cNMP. During WB reperfusion, livers exhibited a blunted cytokine release, regardless of ischemic damage, supporting the potential reconditioning effect of cNMP.

High-Throughput Analysis of Neutrophil Extracellular Trap Levels in Subtypes of People with Type 1 Diabetes.

Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol-myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease.

An adipocentric perspective on the development and progression of non-alcoholic fatty liver disease.

Alongside the liver, white adipose tissue (WAT) is critical in regulating systemic energy homeostasis. Although each organ has its specialised functions, they must work coordinately to regulate whole-body metabolism. Adipose tissues and the liver are relatively resilient and can adapt to an energy surplus by facilitating triglyceride (TG) storage up to a certain threshold level without significant metabolic disturbances. However, lipid storage in WAT beyond a "personalised" adiposity threshold becomes dysfunctional, leading to metabolic inflexibility, progressive inflammation, and aberrant adipokine secretion. Moreover, the failure of adipose tissue to store and mobilise lipids results in systemic knock-on lipid overload, particularly in the liver. Factors contributing to hepatic lipid overload include lipids released from WAT, dietary fat intake, and enhanced de novo lipogenesis. In contrast, extrahepatic mechanisms counteracting toxic hepatic lipid overload entail coordinated compensation through oxidation of surplus fatty acids in brown adipose tissue and storage of fatty acids as TGs in WAT. Failure of these integrated homeostatic mechanisms leads to quantitative increases and qualitative alterations to the lipidome of the liver. Initially, hepatocytes preferentially accumulate TG species leading to a relatively "benign" non-alcoholic fatty liver. However, with time, inflammatory responses ensue, progressing into more severe conditions such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, in some individuals (often without an early prognostic clue). Herein, we highlight the pathogenic importance of obesity-induced "adipose tissue failure", resulting in decreased adipose tissue functionality (i.e. fat storage capacity and metabolic flexibility), in the development and progression of NAFL/NASH.

Patients with a History of Bariatric Surgery Are 8 Years Younger at Presentation with Severe Alcoholic Hepatitis.

PURPOSE: Patients with prior bariatric surgery (BS) are at risk to develop alcohol use disorder (AUD) and alcohol-related liver disease (ALD). Severe alcoholic hepatitis (sAH) is one of the most severe manifestations of ALD with a 28-day mortality of 20-50%. The impact of prior BS on patients presenting with sAH was assessed. METHODS: From 01/2008 to 04/2021, consecutive patients admitted to a tertiary referral center with biopsy-proven sAH were included in a database. RESULTS: One hundred fifty-eight sAH patients of which 28 patients had a history of BS (BS group) were identified. Of this BS group, 24 patients underwent a Roux-en-Y gastric bypass (RYGB), 3 a biliopancreatic diversion, 1 an adjustable gastric band, and no patients a sleeve gastrectomy. The proportion of patients with BS increased threefold over time during the study period. Patients in the BS group were significantly younger at diagnosis of sAH (44.3 years vs 52.4 years), were more frequently female, and had a higher body mass index and a higher grade of steatosis on liver biopsy. The correlation between BS and a younger age at diagnosis remained significant in a multivariate regression analysis. There were no differences in disease severity between both groups. Furthermore, there were no differences in corticosteroid response, 28-day, 90-day, or 1-year survival. CONCLUSION: Prior BS is independently associated with a younger age of presentation with sAH, but is not independently associated with a different disease severity or outcome. These findings support the need for early detection of AUD in patients who underwent BS, in particular RYGB.

Differential Cytokine Levels during Normothermic Kidney Perfusion with Whole Blood- or Red Blood Cell-Based Perfusates-Results of a Scoping Review and Experimental Study.

The ideal composition of the perfusate for normothermic kidney perfusion is unknown, though the perfusate commonly used to perfuse human kidneys contains leukocyte-depleted packed red blood cells (RBC), as this is believed to prevent excessive inflammation. We performed a systematic search identifying 19 articles reporting on cytokine levels during normothermic pig or human kidney perfusion. Cytokine levels varied widely across the reported studies. No direct comparisons of perfusate cytokines during perfusion with RBC or whole blood were performed, and no data on how these levels are influenced by ischemia are available. Therefore, we compared perfusate IL-6, IL-1ß, TNF-α, TGF-ß, IL-10, IL-8, and CCL2 levels during 4 h normothermic pig kidney perfusion with a whole blood- or RBC-based perfusate. Kidneys were exposed to either 1 h of warm or 22 h of cold ischemia. We found no evidence of different perfusate cytokine or gene expression levels in whole blood or RBC perfusions. There was no clear evidence to suggest that cytokine concentrations differ between ischemically injured kidneys and controls. In conclusion, pro-inflammatory and anti-inflammatory cytokines and chemokines are detectable in the perfusate and urine of kidneys undergoing normothermic perfusion. It is unclear how cytokine levels change in different ischemic conditions and whether the use of a leukocyte filter plays a role.

Fluorine MR Imaging Probes Dynamic Migratory Profiles of Perfluorocarbon-Loaded Dendritic Cells After Streptozotocin-Induced Inflammation.

PURPOSE: The pathogenesis of type 1 diabetes (T1D) involves presentation of islet-specific self-antigens by dendritic cells (DCs) to autoreactive T cells, resulting in the destruction of insulin-producing pancreatic beta cells. We aimed to study the dynamic homing of diabetes-prone DCs to the pancreas and nearby organs with and without induction of pancreatic stress in a T1D susceptible model of repeated streptozotocin (STZ) injection. PROCEDURES: In vitro labeling of activated bone marrow-derived DCs (BMDCs) from NOD (Nonobese diabetes) mice was performed using zonyl perfluoro-15-crown-5-ether nanoparticles (ZPFCE-NPs). Internalization of particles was confirmed by confocal microscopy. Two groups of NOD.SCID (nonobese diabetic/severe combined immunodeficiency) mice with (induced by low dose STZ administration) or without pancreatic stress were compared. Diabetogenic BMDCs loaded with BDC2.5 mimotope were pre-labeled with ZPFCE-NPs and adoptively transferred into mice. Longitudinal in vivo fluorine MRI (19F MRI) was performed 24 h, 36 h and 48 h after transfer of BMDCs. For ex vivo quantification of labeled cells, 19F NMR and flow cytometry were performed on dissected tissues to validate in vivo 19F MRI data. RESULTS: In vitro flow cytometry and confocal microscopy confirmed high uptake of nanoparticles in BMDCs during the process of maturation. Migration/homing of activated and ZPFCE-NP- labeled BMDCs to different organs was monitored and quantified longitudinally, showing highest cell density in pancreas at 48-h time-point. Based on 19F MRI, STZ induced mild inflammation in the pancreatic region, as indicated by high accumulation of ZPFCE-NP-labeled BMDCs in the pancreas when compared to the vehicle group. Pancreatic draining lymph nodes showed elevated homing of labeled BMDCs in the vehicle groups in contrast to the STZ group after 72 h. The effect of STZ was confirmed by increased blood glucose levels. CONCLUSION: We showed the potential of 19F MRI for the non-invasive visualization and quantification of migrating immune cells in models for pancreatic inflammation after STZ administration. Without any intrinsic background signal, 19F MRI serves as a highly specific imaging tool to study the migration of diabetic-prone BMDCs in T1D models in vivo. This approach could particularly be of interest for the longitudinal assessment of established or novel anti-inflammatory therapeutic approaches in preclinical models.

Treatment of severe alcoholic hepatitis: A systematic review.

Severe alcoholic hepatitis is the most severe form of alcohol-related liver disease. Corticosteroids remain the first choice of treatment. However, they are only effective in a subset of patients and are associated with an increased infection risk. Furthermore, nonresponders to corticosteroids have a poor prognosis with a mortality of 70% over 6 months. As such, there is a high need for a more personalized use of corticosteroids and the development and identification of alternative therapeutic strategies. In this review, we summarize the recent and ongoing randomized controlled trials concerning the treatment of severe alcoholic hepatitis.

Is there a role for neuregulin 4 in human nonalcoholic fatty liver disease?

BACKGROUND: Neuregulin 4 (Nrg4), a novel adipokine enriched in brown adipose tissue has been observed to negatively regulate de novo hepatic lipogenesis and limit nonalcoholic fatty liver disease (NAFLD) progression to nonalcoholic steatohepatitis (NASH) in rodents. However, the role of Nrg4 in human NAFLD remains unclear to date. We analysed Nrg4 plasma levels and its association with liver disease severity together with the transcriptional profile of the Nrg4 pathway in liver and visceral adipose tissue (VAT) of NAFLD patients. METHODS: Plasma Nrg4 levels were measured in 65 NAFLD patients and 43 healthy controls (HC). Hepatic steatosis and fibrosis were diagnosed and quantified with chemical shift MRI and transient elastography respectively. Furthermore, blood lipid levels, HOMA-IR and systemic pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ) were analysed. Microarray analyses to assess differences in the Nrg4 and its receptor family ErbB pathway in liver and VAT from an independent patient group with biopsy proven NAFL (simple steatosis) (n = 4), NASH (n = 5) and normal liver (n = 6) were performed. RESULTS: Plasma Nrg4 levels were not significantly different between NAFLD patients and HC (p = 0.622). Furthermore, plasma Nrg4 levels did not correlate with the hepatic fat fraction (r = -0.028, p = 0.829) and were not significantly different between NAFLD patients with or without hepatic fibrosis (p = 0.087). Finally, the expression profile of 82 genes related to the Nrg4-ErbB pathway in liver and VAT was not significantly different between NAFL, NASH or obese controls. CONCLUSION: Our study does not support a role for Nrg4 in the pathophysiology of human NAFLD.

A Critical Blimp-1-Dependent IL-10 Regulatory Pathway in T Cells Protects From a Lethal Pro-inflammatory Cytokine Storm During Acute Experimental Trypanosoma brucei Infection.

In many infectious diseases, the immune response operates as a double-edged sword. While required for protective immunity, infection-induced inflammation can be detrimental if it is not properly controlled, causing collateral body damage and potentially leading to death. It is in this context that the potent anti-inflammatory cytokine interleukin-10 (IL-10) is required to dampen the pro-inflammatory immune response that hallmarks trypanosomosis. Effective control of this infection requires not just the action of antibodies specific for the parasite's variable surface glycoprotein (VSG) coat antigens, but also a pro-inflammatory immune response mediated mainly by IFNγ, TNF, and NO. However, strict control of inflammation is mandatory, as IL-10-deficient mice succumb from an unrestrained cytokine storm within 10 days of a Trypanosome brucei infection. The relevant cellular source of IL-10 and the associated molecular mechanisms implicated in its trypanosomosis associated production are poorly understood. Using an IL-10 reporter mouse strain (Vert-X), we demonstrate here that NK cells, CD8+ T cells and CD4+ T cells as well as B cells and plasma cells constitute potential cellular sources of IL-10 within the spleen and liver during acute infection. The IL-10 wave follows peak pro-inflammatory cytokine production, which accompanied the control of peak parasitemia. Similar results were observed following conventional experimental needle infection and physiological infections via T. brucei-infected tsetse flies. Our results show that conditional T cell-specific ablation of the IL-10 regulating Prdm1 gene (encoding for the Blimp-1 transcription factor), leads to an uncontrolled trypanosome-induced pro-inflammatory syndrome like the one observed in infected IL-10-deficient mice. This result indicates that the biological role of IL-10-derived from non-T cells, including NK cells, is of minor importance when considering host survival. The cytokine IL-27 that is also considered to be an IL-10 regulator, did not affect IL-10 production during infection. Together, these data suggest that T. brucei activates a Blimp-1-dependent IL-10 regulatory pathway in T cells that acts as a critical anti-inflammatory rheostat, mandatory for host survival during the acute phase of parasitemia.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling.

OBJECTIVE: Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined. DESIGN: We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rbhigh T-cell transfer model in combination with several genetic mouse models. RESULTS: Anti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rbhigh T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF. CONCLUSION: The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

Longitudinal In Vivo Assessment of Host-Microbe Interactions in a Murine Model of Pulmonary Aspergillosis.

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (19F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of 19F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression.