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Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage-epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium, a model of enteropathogenic and enterohemorrhagic E. coli infection in humans, promoted a strong type 1/IL-22-driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C. rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E. coli and other intestinal pathogens.

Background & aimsLoss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo.MethodsConstitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers.ResultsPartial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice.ConclusionsConstitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.

ObjectivesAlterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria.DesignIBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain.ResultsLoss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease.ConclusionsOur findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

This study aimed to assess the biochemical methane potential (BMP) of microalgal substrates grown on high-strength beverage wastewater as a potential end-use application for biomass. Two microalgal species, Scenedesmus obliquus and Chlorella pyrenoidosa, were cultivated in standard Blue Green-11 (BG-11) media and high-strength beverage wastewater with a COD of around 5,000 mg L−1. A BMP of 185.53 ± 2.66 mL CH4 g−1 VSadded was obtained from Scenedesmus grown in BG-11 media, which increased to 336.35 ± 26.56 mL CH4 g−1 VSadded after being grown in beverage wastewater. Similar increasing patterns were also observed in the case of Chlorella, where the BMP increased from 179.64 ± 4.14 to 245.51 ± 29.35 mL CH4 g−1 VSadded when changing the media from BG-11 to beverage wastewater. After lipid extraction, microalgae substrates yielded higher methane production, with 393.66 ± 41.08 and 421.86 ± 47.52 mL CH4 g−1 VSadded from Scenedesmus and Chlorella, respectively. Theoretical methane estimation from carbohydrate, protein, and lipid contents resulted in overestimating BMP. However, ANOVA statistics highlighted significant differences in experimental methane yields, and the modified Gompertz model validated the predicted BMP with the experimental values, with an R2 above 0.94. Overall, this study outlines the potential for biomethane enhancement from microalgae grown in wastewater, with an extended energy recovery opportunity after lipid extraction. [ABSTRACT FROM AUTHOR]

The localized drug action in tumors to overcome the side effects of chemotherapy has become an impetus for the development of photoactivated chemotherapy (PACT). As potential PACT agents, ruthenium(II) polypyridyl complexes have emerged as efficient photocages for anticancer agents. Bioactive molecules possessing functional groups such as nitrile, thioether, pyridine, imidazole, etc. are often directly attached to the primary coordination sphere of Ru(II) polypyridyl complexes for this purpose. Herein, we propose an alternative design strategy to attach potential anticancer agents lacking these functional groups with Ru(II) polypyridyl complexes through a pyridyl linker moiety. The proposition is, however, a thoughtful extrapolation of a serendipitous crossed aldol reaction that took place between the Ru(II)-coordinated 4-Pyridinecarboxaldehyde (4-PyCHO) and acetone, discovered while the Ru(II)-complex [Ru(ttp)(dppz)(4-PyCHO)]2+ {[1]} [ttp = p-tolyl terpyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, 4-PyCHO = 4-Pyridinecarboxaldehyde] was being purified by silica gel column chromatography with acetone/water/saturated aqueous KNO3 solution as the eluent. The resultant pure aldol product [Ru(ttp)(dppz)(4-PyCHAc)]2+ {[1-Ac]} [4-PyCHAc = aldol modified 4-Pyridinecarboxaldehyde, i.e., 4-hydroxy-4-(pyridin-4-yl)butan-2-one)], was unambiguously characterized by a variety of spectroscopic techniques and X-ray crystallography. Furthermore, a 1H NMR study after 470 nm light irradiation and subsequent ESI-MS analysis revealed that 4-PyCHO could be photo-released from [1-Ac] as its in situ generated aldol adduct 4-PyCHAc. Therefore, this finding serves as a proof-of-concept that provides a simpler alternative design strategy for appending cancer-selective agents having carbonyl groups with α-hydrogens to ruthenium(II) polypyridyl complexes and their photorelease for selective and targeted anticancer chemotherapy. [ABSTRACT FROM AUTHOR]

The removal of nitrogen from wastewater is one of the challenges faced by environmental engineers globally. Autotrophic denitrification is a promising method for treating nitrate (NO3−) -contaminated wastewater because of its relatively low requirement for carbon and lesser sludge yield. Therefore, this study was aimed at investigating the autotrophic denitrification efficiency of two different dual-chamber configurations of microbial fuel cells (MFCs) in order to provide a foundation for building scalable, efficient, and sustainable denitrification systems. Analysis revealed that the maximum current density and power density were 4,327 mA/m3 and 1,441 mW/m3, respectively, in a cube-shaped dual-chamber MFC (C-MFC), whereas these values were 1,452 mA/m3 and 370 mW/m3, respectively, in an H-shaped dual-chamber configuration (H-MFC). In addition, the C-MFC and H-MFC achieved relatively high NO3− reduction rates of up to 28 and 15.6 g/m3/day, respectively. The cathodic coulombic efficiency was observed to be 57% in the C-MFC and 30% in the H-MFC. The average chemical oxygen demand removal efficiency was about 98% for both MFC configurations. High-throughput sequencing analysis revealed significant changes in the microbial community structure under different reactor configurations. The relative abundance of Proteobacteria (34.67%) and Firmicutes (18.59%) at the phylum level were higher in the C-MFC than the H-MFC, signifying that higher current generation can enhance the proliferation and microbial activity of autotrophic denitrifiers. [ABSTRACT FROM AUTHOR]