CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Comparison of Inflammatory Effects in THP-1 Monocytes and Macrophages after Exposure to Metal Ions.

Monocytes and macrophages are the first barrier of the innate immune system, which interact with abrasion and corrosion products, leading to the release of proinflammatory mediators and free reactive molecules. The aim of this study was to understand inflammation-relevant changes in monocytes and macrophages after exposure to corrosion products. To do this, the THP-1 cell line was used to analyze the effects of metal ions simultaneously in monocytes and differentiated macrophages. Cells were stimulated with several concentrations of metal salts (CoCl2, NiCl2, CrCl3 × 6H2O) to analyze viability, gene expression, protein release and ROS production. Untreated cells served as negative controls. While exposure to Cr(3+) did not influence cell viability in both cell types, the highest concentration (500 µM) of Co(2+) and Ni(2+) showed cytotoxic effects mirrored by significantly reduced metabolism, cell number and a concomitant increase of ROS. The release of IL-1ß, IL-8, MCP-1 and M-CSF proteins was mainly affected in macrophages after metal ion exposure (100 µM), indicating a higher impact on pro-inflammatory activity. Our results prove that monocytes and macrophages react very sensitively to corrosion products. High concentrations of bivalent ions lead to cell death, while lower concentrations trigger the release of inflammatory mediators, mainly in macrophages.

Analysis of Cellular Activity Short-Term Exposure to Cobalt and Chromium Ions in Mature Human Osteoblasts.

In aseptic loosening of endoprosthetic implants, metal particles, as well as their corrosion products, have been shown to elicit a biological response. Due to different metal alloy components, the response may vary depending on the nature of the released corrosion product. Our study aimed to compare the biological effects of different ions released from metal alloys. In order to mimic the corrosion products, different metal salts (CoCl2, NiCl2 and CrCl3 × 6H2O) were dissolved and allowed to equilibrate. Human osteoblasts were incubated with concentrations of 10 µM to 500 µM metal salt solutions under cell culture conditions, whereas untreated cells served as negative controls. Cells exposed to CoCr28Mo6 particles served as positive controls. The cell activity and expression of osteogenic differentiation and pro-osteolytic mediators were determined. Osteoblastic activity revealed concentration- and material-dependent influences. Collagen 1 synthesis was reduced after treatment with Co(2+) and Ni(2+). Additionally, exposure to these ions (500 µM) resulted in significantly reduced OPG protein synthesis, whereas RANKL as well as IL-6 and IL-8 secretion were increased. TLR4 mRNA was significantly induced by Co(2+) and CoCr28Mo6 particles. The results demonstrate the pro-osteolytic capacity of metal ions in osteoblasts. Compared to CoCr28Mo6 particles, the results indicated that metal ions intervene much earlier in inflammatory processes.

Single-sex infection with female Schistosoma mansoni cercariae mitigates hepatic fibrosis after secondary infection.

BACKGROUND: Infection with Schistosoma spp. affects more than 258 million people worldwide. Current treatment strategies are mainly based on the anthelmintic Praziquantel, which is effective against adult worms but neither prevents re-infection nor cures severe liver damage. The best long-term strategy to control schistosomiasis may be to develop an immunization. Therefore, we designed a two-step Schistosoma mansoni infection model to study the immune-stimulating effect of a primary infection with either male or female cercariae, measured on the basis of TH1/TH2-response, granuloma size and hepatic fibrosis after a secondary bisexual S. mansoni challenge. METHODOLOGY/PRINCIPLE FINDINGS: As a first step, mice were infected with exclusively female, exclusively male, or a mixture of male and female S. mansoni cercariae. 11 weeks later they were secondarily infected with male and female S. mansoni cercariae. At week 19, infection burden, granuloma size, collagen deposition, serum cytokine profiles and the expression of inflammatory genes were analyzed. Mice initially infected with female S. mansoni cercariae displayed smaller hepatic granulomas, livers and spleens, less hepatic fibrosis and higher expression of Ctla4. In contrast, a prior infection with male or male and female S. mansoni did not mitigate disease progression after a bisexual challenge. CONCLUSIONS/SIGNIFICANCE: Our findings provide evidence that an immunization against S. mansoni is achievable by exploiting gender-specific differences between schistosomes.

High Pneumocystis jirovecii colonization rate among haemodialysis patients.

Haemodialysis patients have been found to have an increased risk of developing Pneumocystis pneumonia (PcP) compared to the control population. To the best of our knowledge, no data are available on pulmonary colonization with Pneumocystis jirovecii in haemodialysis patients; therefore, the aim of this study was to determine the prevalence of pulmonary colonization with P. jirovecii in haemodialysis patients, and to find the related risk factors. Induced sputa of 62 haemodialysis patients were investigated using quantitative polymerase chain reaction for the presence of P. jirovecii. 20.9% of the patients were colonized with P. jirovecii and 46.2% of whom had CD4 cell counts below 400/µl. There was no significant correlation between colonization and time on dialysis treatment. As haemodialysis patients seem to be at higher risk of PcP than the general population, doctors should be aware of the high rate of P. jirovecii colonization amongst them. Furthermore, colonized patients remain a potential source of transmission of P. jirovecii to other patients or to health care workers.

24-nor-ursodeoxycholic acid ameliorates inflammatory response and liver fibrosis in a murine model of hepatic schistosomiasis.

BACKGROUND & AIMS: Intrahepatic granuloma formation and fibrosis characterize the pathological features of Schistosoma mansoni infection. Based on previously observed substantial anti-fibrotic effects of 24-nor-ursodeoxycholic acid (norUDCA) in Abcb4/Mdr2(-/-) mice with cholestatic liver injury and biliary fibrosis, we hypothesized that norUDCA improves inflammation-driven liver fibrosis in S. mansoni infection. METHODS: Adult NMRI mice were infected with 50 S. mansoni cercariae and after 12 weeks received either norUDCA- or ursodeoxycholic acid (UDCA)-enriched diet (0.5% wt/wt) for 4 weeks. Bile acid effects on liver histology, serum biochemistry, key regulatory cytokines, hepatic hydroxyproline content as well as granuloma formation were compared to naive mice and infected controls. In addition, effects of norUDCA on primary T-cell activation/proliferation and maturation of the antigen-presenting-cells (dendritic cells, macrophages) were determined in vitro. RESULTS: UDCA as well as norUDCA attenuated the inflammatory response in livers of S. mansoni infected mice, but exclusively norUDCA changed cellular composition and reduced size of hepatic granulomas as well as TH2-mediated hepatic fibrosis in vivo. Moreover, norUDCA affected surface expression level of major histocompatibility complex (MHC) class II of macrophages and dendritic cells as well as activation/proliferation of T-lymphocytes in vitro, whereas UDCA had no effect. CONCLUSIONS: This study demonstrates pronounced anti-inflammatory and anti-fibrotic effects of norUDCA compared to UDCA in S. mansoni induced liver injury, and indicates that norUDCA directly represses antigen presentation of antigen presenting cells and subsequent T-cell activation in vitro. Therefore, norUDCA represents a promising drug for the treatment of this important cause of liver fibrosis.

Pneumocystis jirovecii colonization among renal transplant recipients.

AIM: Renal transplant recipients are at risk of developing Pneumocystis pneumonia (PcP), especially in the first 2 years after transplantation, with a mortality rate of up to 50%. No data are available on pulmonary colonization with Pneumocystis jirovecii in renal transplant recipients. The aim of this study was to determine the prevalence of pulmonary colonization with Pneumocystis jirovecii in renal transplant recipients and to find related risk factors. METHODS: We investigated the induced sputa of 70 renal transplant recipients for the presence of Pneumocystis jirovecii using nested polymerase chain reaction. RESULTS: Thirteen of 70 patients (18.6%) were colonized with Pneumocystis jirovecii. There was no significant correlation between colonization and immunosuppressive medication or regimens. However, colonized subjects had undergone transplantation longer ago than non-colonized subjects. 30.8% of those whose transplantation had taken place more than 8 years previously were colonized, in contrast to 11.4% of those whose transplantation had taken place less than 8 years ago (P = 0.059; odds ratio = 3.467, 95% confidence interval = 0.99-12.09). CONCLUSION: Most cases of Pneumocystis colonization were detected in those patients where renal transplantion had taken place more than 2 years previously. As most PcP cases occur within the first 2 years of transplantation, colonization does not seem to play a role in the development of acute PcP in this period. Though Pneumocystis pneumonia is likely to be a newly acquired infection in the first 2 years after transplantation, colonized patients remain a potential source of transmission of Pneumocystis jirovecii.

The lack of memory B cells including T cell independent IgM+ IgD+ memory B cells in chronic graft-versus host disease is associated with susceptibility to infection.

The chronic graft-versus host disease (cGVHD) is associated with a perturbed B cell homeostasis and an increased infection rate. Aiming to determine the impact of lymphocyte subsets on cGVHD, blood samples from 98 patients at least 100 days following allogeneic haematopoietic stem cell transplantation (median 1066 days) were analyzed, serum levels of immunoglobulins measured and the incidence of severe infections retrospectively documented. Absolute CD19(+) B cell counts, including counts of immature (CD10(+) CD38(++) CD20(+) IgM(++)) and transitional (CD10(-) CD38(++) CD20(+) IgM(++)) as well as class switched memory (CD19(+) CD27(+) IgM(-) IgD(-)) B cells in patients with active cGVHD (n = 52) were significantly decreased as compared to those with inactive (n = 18) or without cGVHD (n = 28). In addition, nonclass switched IgM(+) memory B cells (CD19(+) CD27(+) IgM(+) IgD(+)) were absent in patients with cGVHD, but not in patients with inactive (0.4 × 10(6) /l) or without (1.7 × 10(6) /l) cGVHD (both P < 0.001). In line with these results we found significantly decreased lgG levels in patients with cGVHD, which was associated with a significantly higher rate of severe infections in cGVHD patients. Our data underline the close association of diminished B cell counts with cGVHD and the onset of severe infections. The lack of IgM(+) memory B cells in patients with cGVHD may indicate functional asplenia.

The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles.

Aseptic loosening in total hip replacement is mainly caused by wear particles inducing inflammation and osteolysis. Wear can be a consequence of micromotions at the interface between implant and bone cement. Due to complex cellular interactions, different mediators (e.g. cytokines, proteinases) are released, which can promote osteolytic processes in the periprosthetic tissue followed by loosening of the implant. Furthermore, a reduced matrix synthesis and an induced apoptosis rate can be observed. The purpose of this study was to evaluate to what extent human primary osteoblasts exposed to wear particles are involved in the osteolysis. The viability, the secretion of collagen and collagenases and the variety of released cytokines after particle exposure was examined. Therefore, human osteoblasts were incubated with particles experimentally generated in the interface between hip stems with rough and smooth surface finishings as well as different material compositions (Ti-6Al-7Nb, Co-28Cr-6Mo and 316L) and bone cement mantle made of Palacos R containing zirconium oxide particles. Commercially pure titanium particles, titanium oxide, polymethylmethacrylate and particulate zirconium oxide were used as references. The results revealed distinct effects on the cytokine release of human osteoblasts towards particulate debris. Thereby, human osteoblasts released increased levels of interleukine (IL)-6 and IL-8 after treatment with metallic wear particles. The expression of VEGF was slightly induced by all particle entities at lower concentrations. Apoptotic rates were enhanced for osteoblasts exposed to all the tested particles. Furthermore, the de novo synthesis of type 1 collagen was reduced and the expression of the matrix metalloproteinase (MMP)-1 was considerably increased. However, wear particles of Co-28Cr-6Mo stems seemed to be more aggressive, whereas particles derived from stainless steel stems caused less adverse cellular reaction. Among the reference particles, which caused less altered reactions in the metabolism of osteoblasts in general, ZrO2 can be assumed as the material with the smallest cell biological effects.

Differentiation capacity of human chondrocytes embedded in alginate matrix.

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco?s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.

The impact of HLA-DRB alleles on the subclass titres of antibodies against citrullinated peptides.

OBJECTIVES: The association between HLA-DR haplotypes and RA have been well established. However, the molecular mechanisms of how HLA mediates susceptibility and/or progression of the disease remain elusive. We therefore turned to the RA-specific antibodies directed against citrullinated peptide antigens (ACPAs) and investigated the association between HLA-DRB1 shared epitope (SE) alleles and the IgG subclass titres of cyclic citrullinated peptide (CCP)- and mutated citrullinated vimentin (MCV)-specific antibodies. METHODS: One hundred and twenty-seven RA patients were typed for their HLA-DRB1 haplotypes applying low resolution and alleles potentially carrying the SE were sequenced. All patients' sera were analysed by ELISA for the presence of ACPA and 77 patients positive for CCP-specific antibodies were further analysed for the respective IgG subclasses. Subclass titres were then correlated to the presence of a SE. Finally, all patients were screened for the HLA-DRB4-associated splice variant. RESULTS: We found a gene dosage effect of the HLA-DRB1*04-associated SE on both the MCV- and CCP-specific IgG3 levels. The HLA-DRB4-associated splice variant accumulates in ACPA-negative RA patients. CONCLUSIONS: Both the dose-dependent increase in IgG3 among ACPA and the accumulation of the splice variant in ACPA-negative patients imply differential expression of the HLA alleles as the mechanism contributing to the susceptibility and/or disease progression of RA. The preponderance of IgG3 hints at a skewing towards a Th1 response and is reminiscent of increased signal strengths at the immunological synapse. Likewise, the abrogation of HLA-DRB4 expression due to the splice variant reduces the signal strength and seems to protect from ACPA development.

The anti-mutated citrullinated vimentin response classifies patients with rheumatoid arthritis into broad and narrow responders.

OBJECTIVE: Autoantibodies against citrullinated peptide antigens (ACPA) are routinely determined to diagnose rheumatoid arthritis (RA) and are predictive of a more severe course of the disease. We here set out to address an involvement of ACPA in the pathogenesis of RA and investigated the recognition pattern of antibodies against 2 citrullinated antigens in more detail. METHODS: The sera of 77 patients fulfilling the American College of Rheumatology criteria for RA were analyzed for subclass titers of anti-mutated citrullinated vimentin (MCV) and anticyclic citrullinated peptide (CCP) antibodies by combining subclass specific detection antibodies with commercially available CCP and MCV ELISA plates. Cross-reactivities between anti-MCV and anti-CCP antibodies were detected using a sequential ELISA system. RESULTS: IgG1, IgG3, and IgG4 titers among anti-MCV and anti-CCP antibodies correlated significantly. Cross-reactivity of MCV-specific antibodies against CCP could be detected in 8 of 16 patients' sera; however, cross-binding of MCV-specific IgG4 was weaker compared to total IgG. CONCLUSION: The inherent capacity of IgG4 to exchange F(ab) arms provides insight into the anti-MCV antibody diversity and suggests a classification of ACPA positive patients into broad and narrow responders.