Serum amyloid alpha 1-2 are not required for liver inflammation in the 4T1 murine breast cancer model

がんに起因して起こる宿主の肝臓の急性期応答と炎症 --血清アミロイドαは乳がんモデルにおける肝臓の炎症の原因ではない--. 京都大学プレスリリース. 2023-02-06.Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause inflammation in various host organs. Despite the well-known coincidence of acute phase response and inflammation, the direct roles of SAA proteins in inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in liver inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. Consistently, 4T1-induced gene expression changes in bone marrow do not require Saa1-2. This study clarifies the negligible contribution of SAA1-2 proteins in liver inflammation in the 4T1 breast cancer model

Murine breast cancers disorganize the liver transcriptome in a zonated manner

がんが宿主の臓器に及ぼす悪影響を捉えた --がんをもつ個体における「肝機能の空間的制御」の破綻--. 京都大学プレスリリース. 2023-02-01.The spatially organized gene expression program within the liver specifies hepatocyte functions according to their relative distances to the bloodstream (i.e., zonation), contributing to liver homeostasis. Despite the knowledge that solid cancers remotely disrupt liver homeostasis, it remains unexplored whether solid cancers affect liver zonation. Here, using spatial transcriptomics, we thoroughly investigate the abundance and zonation of hepatic genes in cancer-bearing mice. We find that breast cancers affect liver zonation in various distinct manners depending on biological pathways. Aspartate metabolism and triglyceride catabolic processes retain relatively intact zonation patterns, but the zonation of xenobiotic catabolic process genes exhibits a strong disruption. The acute phase response is induced in zonated manners. Furthermore, we demonstrate that breast cancers activate innate immune cells in particular neutrophils in distinct zonated manners, rather than in a uniform fashion within the liver. Collectively, breast cancers disorganize hepatic transcriptomes in zonated manners, thereby disrupting zonated functions of the liver

Distinct requirements for the maintenance and establishment of mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) that maintain a sustainable pluripotent state are derived from the inner cell mass (ICM) of blastocysts, in which pluripotency is lost during differentiation in vivo. It is unclear when and how the ability to maintain pluripotency is acquired during the derivation of ESCs. We analyzed the required culture condition for the maintenance and establishment of ESCs in detail. Even at low concentration of the GSK3β inhibitor and LIF (LowGiL), the expression levels of pluripotency markers and the chimera-producing ability of the cells were comparable with those of ESCs cultured in the presence of both inhibitors and LIF (2iL). However, blastocysts underwent spontaneous differentiation, and ESCs were not established under LowGiL condition. Time-course analysis showed that 2iL condition for three days from the initiation of culture was sufficient for the acquisition of permanent pluripotency. Although X chromosome-linked pluripotent genes were significantly up-regulated during the culture of both male and female blastocysts in 2iL condition, no such up-regulation was observed in LowGiL condition. In conclusion, 2iL-dependent activation of these X-linked genes at the earliest phase of ESC derivation is one of the molecular bases for the acquisition of permanent pluripotency. Keywords: Embryonic stem cells, Pluripotency, Gsk3β inhibitor, MEK inhibitor, X-chromosome-linked gene

Correction: Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

[This corrects the article DOI: 10.1371/journal.pone.0154098.]

GST-HsaNR2-3-2 inhibits hemagglutination of human erythrocytes by <i>S</i>. <i>gordonii</i> DL1.

<p>(A) Schematic drawing of GST fusion proteins. NR1 and NR2, non-repetitive regions 1 and 2 (respectively); SR1 and SR2, serine-rich repeat regions 1 and 2 (respectively); CWAD, cell wall-anchoring domain. (B) Hemagglutination of human erythrocytes by <i>S</i>. <i>gordonii</i> DL1. 0.3% human erythrocytes were incubated with serial dilutions of GST-fused HsaNR2 mutant proteins in the presence or absence of <i>S</i>. <i>gordonii</i> DL1.</p

Binding of GST-HsaNR2 mutant proteins to different oligosaccharide structures.

<p>The indicated amount of GST or GST-HsaNR2 wild type (WT) or mutant (R340N and R365N) proteins were immobilized in microtiter wells, and the binding of biotinylated Neu5Acα2-3Gal^®1-4GlcNAc (3’-sialyllactose) (A) and Neu5Acα2-3Gal^®1-3GalNAc (sialyl-T antigen) (B) to each protein was detected using peroxidase-conjugated streptavidin along with a chromogenic peroxidase substrate. Binding is expressed as the mean ± standard deviation from the results of six independent experiments.</p

Mutation of Arg340 and Arg365 residues abolishes platelet aggregation activity.

<p>Washed human platelets were incubated (for 30 minutes at room temperature) with 0.33 mg/ml of GST, GST-HsaNR2WT, or GST-HsaNR2 proteins harboring R340N or R365N substitutions. Percent aggregation of platelets was determined spectrophotometrically. The mean numbers of percent aggregation and standard deviations were determined by six independent experiments. Statistical differences in the means of obtained values were evaluated by an unpaired <i>t</i>-test (* <i>P</i> < 10^-5).</p

Essential role of Hsa Arg340 and Arg365 in adhesion to erythrocytes and platelets.

<p>(A) Expression of wild-type and mutant Hsa proteins on the <i>S</i>. <i>gordonii</i> cell surface. Peptidoglycan-linked proteins were extracted from bacterial cell surface and analyzed by western blotting using anti-Hsa or anti-DL1 antibody. The position of the molecular mass marker is indicated on the left in kilodaltons. (B) Hemagglutination of human erythrocytes by <i>S</i>. <i>gordonii</i>. 0.3% human erythrocytes were incubated (overnight at 4°C) with serial dilutions of <i>S</i>. <i>gordonii</i> DL1 or derivative strains. (C) Platelet aggregation activity of <i>S</i>. <i>gordonii</i>. Washed human platelets were incubated (for 30 minutes at room temperature) with <i>S</i>. <i>gordonii</i> DL1 or derivative strains. Percent aggregation of platelets was determined spectrophotometrically. The mean numbers of percent aggregation and standard deviations were determined by five independent experiments. Statistical differences in the means of obtained values were evaluated by an unpaired <i>t</i>-test (**<i>P</i> < 0.0001).</p

Attachment of <i>S</i>. <i>gordonii</i> DL1 to human leukocytes.

<p>(A) Human leukocytes (5× 10⁵ cells) were incubated with <i>S</i>. <i>gordonii</i> DL1 (a, b, c, and d), CM100 (e, f, g, and h), CM100 (WT) (i, j, k, and l), CM100 (R340N) (m, n, o, and p), or CM100 (R365N) (g, r, s, and t) (5× 10⁷ cells) for 2 h at 37°C. Bacterial binding was determined microscopically using Wright-Giemsa staining. Arrowheads indicate phagosomes that appear to contain intact bacterial cells. Original magnification, × 1000. Scale bar = 10 μm. (B) The means and standard deviations of bacteria cell numbers bound to randomly selected neutrophils, eosinophils, monocytes, or lymphocytes (n = 30) are indicated. Statistical differences in the means of obtained values were evaluated by an unpaired <i>t</i>-test (*** <i>P</i> < 10^-14).</p