Myeloid A20 is critical for alternative macrophage polarization and type-2 immune-mediated helminth resistance.

Background: Protective immunity against intestinal helminths requires induction of robust type-2 immunity orchestrated by various cellular and soluble effectors which promote goblet cell hyperplasia, mucus production, epithelial proliferation, and smooth muscle contractions to expel worms and re-establish immune homeostasis. Conversely, defects in type-2 immunity result in ineffective helminth clearance, persistent infection, and inflammation. Macrophages are highly plastic cells that acquire an alternatively activated state during helminth infection, but they were previously shown to be dispensable for resistance to Trichuris muris infection. Methods: We use the in vivo mouse model A20myel-KO, characterized by the deletion of the potent anti-inflammatory factor A20 (TNFAIP3) specifically in the myeloid cells, the excessive type-1 cytokine production, and the development of spontaneous arthritis. We infect A20myel-KO mice with the gastrointestinal helminth Trichuris muris and we analyzed the innate and adaptive responses. We performed RNA sequencing on sorted myeloid cells to investigate the role of A20 on macrophage polarization and type-2 immunity. Moreover, we assess in A20myel-KO mice the pharmacological inhibition of type-1 cytokine pathways on helminth clearance and the infection with Salmonella typhimurium. Results: We show that proper macrophage polarization is essential for helminth clearance, and we identify A20 as an essential myeloid factor for the induction of type-2 immune responses against Trichuris muris. A20myel-KO mice are characterized by persistent Trichuris muris infection and intestinal inflammation. Myeloid A20 deficiency induces strong classical macrophage polarization which impedes anti-helminth type-2 immune activation; however, it promotes detrimental Th1/Th17 responses. Antibody-mediated neutralization of the type-1 cytokines IFN-γ, IL-18, and IL-12 prevents myeloid-orchestrated Th1 polarization and re-establishes type-2-mediated protective immunity against T. muris in A20myel-KO mice. In contrast, the strong Th1-biased immunity in A20myel-KO mice offers protection against Salmonella typhimurium infection. Conclusions: We hereby identify A20 as a critical myeloid factor for correct macrophage polarization and appropriate adaptive mucosal immunity in response to helminth and enteric bacterial infection.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

The multi-omics single-cell landscape of sinus mucosa in uncontrolled severe chronic rhinosinusitis with nasal polyps.

Uncontrolled severe chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with elevated levels of type 2 inflammatory cytokines and raised immunoglobulin concentrations in nasal polyp tissue. By using single-cell RNA sequencing, transcriptomics, surface proteomics, and T cell and B cell receptor sequencing, we found the predominant cell types in nasal polyps were shifted from epithelial and mesenchymal cells to inflammatory cells compared to nasal mucosa from healthy controls. Broad expansions of CD4 T effector memory cells, CD4 tissue-resident memory T cells, CD8 T effector memory cells and all subtypes of B cells in nasal polyp tissues. The T and B cell receptor repertoires were skewed in NP. This study highlights the deviated immune response and remodeling mechanisms that contribute to the pathogenesis of uncontrolled severe CRSwNP. CLINICAL IMPLICATIONS: We identified differences in the cellular compositions, transcriptomes, proteomes, and deviations in the immune profiles of T cell and B cell receptors as well as alterations in the intercellular communications in uncontrolled severe CRSwNP patients versus healthy controls, which might help to define potential therapeutic targets in the future.

Genetic Responses of Metabolically Active Limnospira indica Strain PCC 8005 Exposed to γ-Radiation during Its Lifecycle.

Two morphotypes of the cyanobacterial Limnospira indica (formerly Arthrospira sp.) strain PCC 8005, denoted as P2 (straight trichomes) and P6 (helical trichomes), were subjected to chronic gamma radiation from spent nuclear fuel (SNF) rods at a dose rate of ca. 80 Gy·h-1 for one mass doubling period (approximately 3 days) under continuous light with photoautotrophic metabolism fully active. Samples were taken for post-irradiation growth recovery and RNA-Seq transcriptional analysis at time intervals of 15, 40, and 71.5 h corresponding to cumulative doses of ca. 1450, 3200, and 5700 Gy, respectively. Both morphotypes, which were previously reported by us to display different antioxidant capacities and differ at the genomic level in 168 SNPs, 48 indels and 4 large insertions, recovered equally well from 1450 and 3200 Gy. However, while the P2 straight type recovered from 5700 Gy by regaining normal growth within 6 days, the P6 helical type took about 13 days to recover from this dose, indicating differences in their radiation tolerance and response. To investigate these differences, P2 and P6 cells exposed to the intermediate dose of gamma radiation (3200 Gy) were analyzed for differential gene expression by RNA-Seq analysis. Prior to batch normalization, a total of 1553 genes (887 and 666 of P2 and P6, respectively, with 352 genes in common) were selected based on a two-fold change in expression and a false discovery rate FDR smaller or equal to 0.05. About 85% of these 1553 genes encoded products of yet unknown function. Of the 229 remaining genes, 171 had a defined function while 58 genes were transcribed into non-coding RNA including 21 tRNAs (all downregulated). Batch normalization resulted in 660 differentially expressed genes with 98 having a function and 32 encoding RNA. From PCC 8005-P2 and PCC 8005-P6 expression patterns, it emerges that although the cellular routes used by the two substrains to cope with ionizing radiation do overlap to a large extent, both strains displayed a distinct preference of priorities.

Characterization of the inflammatory microenvironment and hepatic macrophage subsets in experimental hepatocellular carcinoma models.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)- and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

Murine induced pluripotent stem cell-derived neuroimmune cell culture models emphasize opposite immune-effector functions of interleukin 13-primed microglia and macrophages in terms of neuroimmune toxicity.

Cellular models of induced pluripotent stem cell (iPSC)-derived microglia and macrophages are an emerging toolbox to investigate neuroinflammation in vitro. We previously demonstrated that murine iPSC-microglia and iPSC-macrophages display phenotypical activation properties highly comparable to microglia and macrophages in vivo. Here we extended the characterization of iPSC-microglia and iPSC-macrophages with the analysis of their transcriptome profile. Next, these cellular models were employed to evaluate neuroimmune toxicity in vitro and to investigate the immune-modulatory properties of interleukin 13 (IL13), a cytokine known for its ability to protect against neuroinflammation-induced pathology by modulating microglia and macrophage activation. iPSC-microglia and iPSC-macrophages, in co-culture with astrocyte-committed neural stem cells (NSC), were (pre)treated with IL13 and stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ), to assess how IL13 modulates their inflammatory response. Additionally, the use of luciferase-expressing NSC (Luc-NSC) allowed real-time monitoring of immune-mediated neurotoxicity. Despite the known anti-inflammatory properties of IL13, iPSC-microglia primed with IL13 before LPS + IFNγ stimulation significantly increased NO secretion. This was associated with a marked reduction of the luminescence signal produced by Luc-NSC. Interestingly, we observed that IL13 signaling has a divergent functional outcome in microglia as compared to macrophages, as for the latter no major alterations in NO release and Luc-NSC viability were observed upon IL13 (pre)treatment. Finally, the striking IL13-induced upregulation of NO secretion by microglia under pro-inflammatory conditions was confirmed in vivo, where intracerebral delivery of IL13 increased inducible nitric oxide synthase mRNA expression. Concluding, we applied iPSC-derived neuroimmune cell culture models to identify distinct neuroimmune (toxicity) responses of microglia and macrophages to IL13-based immune modulation.

Neuromagnetic evoked responses to complex motions are greatest for expansion.

We analysed evoked magnetic responses to moving random dot stimuli, initially using a 19-channel magnetoencephalography (MEG) system, and subsequently using a 151-channel MEG system. Random dot displays were used to construct complex motion sequences, which we refer to as expansion, contraction, deformation, and rotation. We also investigated lateral translation and a condition in which the directions of the dots were randomised. In all stimulus conditions, the dots were first stationary, then traveled for a brief period (317 s or 542 ms), and were then stationary again. In all conditions, evoked magnetic responses were observed with a widespread bilateral distribution over the observers' heads. Initial recordings revealed a substantially larger evoked magnetic response to the expansion condition than the other conditions. In a revised study, we used a 151-channel MEG system and two stimulus diameters (9.3 and 48 deg), the smaller comparable with the first experiment. The responses were analysed using a nonparametric approach and confirmed our initial observations. In a third study, speed gradients were removed and a new design permitted direct comparisons between motion conditions. The results from all three experiments are consistent with the greater ecological validity of the expansion stimulus.