Cancer metastasis to sentinel lymph nodes (LNs) is often the first marker of potential disease progression. Although it is recognized that tumor-induced lymphangiogenesis facilitates metastasis into LNs in murine models, tumor-induced alterations in human lymphatic vessels remain obscure. Here we use single-cell RNA sequencing and high-resolution spatial transcriptomics to profile lymphatic endothelial cell (LEC) subsets in paired metastatic and non-metastatic LNs obtained from female patients with treatment-naïve breast cancer. Tumor metastasis decreases immunoregulatory LEC subsets, such as PD-L1+ subcapsular sinus LECs, while inducing an increase in capillary-like CD200+ HEY1+ LECs. Matrix Gla protein (MGP) is the most upregulated gene in metastatic LN LECs, and its expression on LECs is TGF-β and VEGF dependent. Upregulated MGP promotes cancer cell adhesion to LN lymphatics. Thus, breast cancer cell metastasis to LNs remodels LEC subsets in human LNs and escalates MGP expression, potentially facilitating cancer cell dissemination through the lymphatic system.

Supplementary Figure S3. Clever-1 is present in rabbit lymphatics but the lymphoma cell line KCA used in the homing experiments lacks its expression.

Supplementary Figure 3 from Vascular Adhesion Protein-1 Enhances Tumor Growth by Supporting Recruitment of Gr-1+CD11b+ Myeloid Cells into Tumors

Supplementary Table S1. Relative intensity of SPARC staining

Background The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). The use of intravenous Interferon beta (IFN β) for the treatment of ARDS was recently tested in a phase III ARDS trial (INTEREST), in which more than half of the patients simultaneously received glucocorticoids. Trial results showed deleterious effects of glucocorticoids when administered together with IFN β, and therefore, we aimed at finding the reason behind this. Methods We first sequenced the genes encoding the IFN α/β receptor of the patients, who participated in the INTEREST study (ClinicalTrials.gov Identifier: NCT02622724 , November 24, 2015) in which the patients were randomized to receive an intravenous injection of IFN β-1a (144 patients) or placebo (152 patients). Genetic background was analyzed against clinical outcome, concomitant medication, and pro-inflammatory cytokine levels. Thereafter, we tested the influence of the genetic background on IFN α/β receptor expression in lung organ cultures and whether, it has any effect on transcription factors STAT1 and STAT2 involved in IFN signaling. Results We found a novel disease association of a SNP rs9984273, which is situated in the interferon α/β receptor subunit 2 (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, more rapid decrease of IFN γ and interleukin-6 (IL-6) levels and better outcome in IFN β treated patients with ARDS, while the major allele associates with a poor outcome especially under concomitant IFN β and glucocorticoid treatment. Moreover, the minor allele of rs9984273 associates with a less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database. Conclusions The distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signaling and glucocorticoids. Graphical abstract

Supplementary Figure S4. B16 melanoma and EL-4 cells are Clever-1 negative.

Supplementary Methods, Figure Legends 1-4 from Vascular Adhesion Protein-1 Enhances Tumor Growth by Supporting Recruitment of Gr-1+CD11b+ Myeloid Cells into Tumors

Supplementary Materials and Methods, Supplementary Figure 1-6 Legends. Materials and Methods, References, Figure 1-6 Legends

Supplementary Table. Phosphorylation profiles of key protein kinases in control and adenosine-treated PC-3 and MDA-MB-231 cells.

Supplementary Figure S5. Density of MRC positive macrophages is not dependent on tumor size and tumor cells are able to induce Clever-1 expression.

Supplementary Figure S2. Genotyping of different Clever-1 targeted mice

Supplementary Figure S3. Intracellular nucleotide and nucleoside levels in control and adenosine-treated PC-3 cells.

The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). Here we report a novel disease association of a SNP rs9984273, which is situated in the interferon alpha/beta receptor (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, lower IFN-gamma and IL-6 levels and less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database, and better outcome in interferon (IFN) beta treated patients with ARDS. Thus, the distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signalling and glucocorticoids.One-Sentence Summary Single nucleotide polymorphism in interferon receptor contributes to corticosteroid response and outcome in ARDS and COVID-19### Competing Interest StatementJJ is an employee of Faron Pharmaceuticals, JJ, MH and SJ own stocks of Faron Pharmaceuticals### Funding StatementThis study was supported by the EU FP7 program, Traumakine, the Finnish Academy, the Jane and Aatos Erkko Foundation and the Sigrid Juselius Foundation### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics connected to the clinical trials ([NCT00789685][1] and [NCT02622724][2]) has been thoroughly presented in references 18 and 20 (Bellingan et al, Lancet Respiratory Medicine 2014 and Ranieri et al, JAMA 2020). The lung specimens were obtained under permission of the ethics authorities of Turku University Hospital (Finland).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present work are contained in the manuscript. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00789685&atom=%2Fmedrxiv%2Fearly%2F2022%2F03%2F13%2F2022.03.10.22272123.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02622724&atom=%2Fmedrxiv%2Fearly%2F2022%2F03%2F13%2F2022.03.10.22272123.atom

Clever-1 also known as Stabilin-1 and FEEL-1 is a scavenger molecule expressed on a subpopulation of anti-inflammatory macrophages and lymphatic endothelial cells (LECs). However, its role in regulating dendritic cell (DC) trafficking and subsequent effects on immunity have remained unexplored. In this study, we demonstrate that DC trafficking from the skin into the draining lymph nodes is compromised in the absence of Clever-1. By adoptive transfer approaches we further show that the poor trafficking is due to the impaired entrance of DCs into afferent lymphatics. Despite this, injections of ovalbumin-loaded DCs into the footpads induced a stronger proliferative response of OT II T cells in the draining lymph nodes. This could be explained by the increased MHC II expression on DCs and a less tolerogenic phenotype of LECs in lymph nodes of Clever-1 knockout mice. Thus, although fewer DCs reach the nodes, they are more active in creating antigen-specific immune responses. This suggests that the DCs migrating to the draining lymph node within Clever-1 positive lymphatics experience immunosuppressive interactions with LECs. In conclusion, besides being a trafficking molecule on lymphatic vasculature Clever-1 is immunosuppressive towards migrating DCs and thus, regulates the magnitude of immune responses created by incoming DCs in the draining lymph nodes.

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer’s patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve B cells (CD19 + /CD62L hi /CD44 lo ) in Peyer’s patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer’s patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer’s patches.

Macrophages, which are highly diverse in different tissues, play a complex and vital role in tissue development, homeostasis, and inflammation. The origin and heterogeneity of tissue‐resident monocytes and macrophages in ovaries remains unknown. Here we identify three tissue‐resident monocyte populations and five macrophage populations in the adult ovaries using high‐dimensional single cell mass cytometry. Ontogenic analyses using cell fate mapping models and cell depletion experiments revealed the infiltration of ovaries by both yolk sac and fetal liver‐derived macrophages already during the embryonic development. Moreover, we found that both embryonic and bone marrow‐derived macrophages contribute to the distinct ovarian macrophage subpopulations in the adults. These assays also showed that fetal‐derived MHC II‐negative macrophages differentiate postnatally in the maturing ovary to MHC II‐positive cells. Our analyses further unraveled that the developmentally distinct macrophage types share overlapping distribution and scavenging function in the ovaries under homeostatic conditions. In conclusion, we report here the first comprehensive analyses of ovarian monocytes and macrophages. In addition, we show that the mechanisms controlling monocyte immigration, the phenotype of different pools of interstitial macrophages, and the interconversion capacity of fetal‐derived macrophages in ovaries are remarkably different from those seen in other tissue niches.

Importance Acute respiratory distress syndrome (ARDS) is associated with high mortality. Interferon (IFN) β-1a may prevent the underlying event of vascular leakage. Objective To determine the efficacy and adverse events of IFN-β-1a in patients with moderate to severe ARDS. Design, Setting, and Participants Multicenter, randomized, double-blind, parallel-group trial conducted at 74 intensive care units in 8 European countries (December 2015-December 2017) that included 301 adults with moderate to severe ARDS according to the Berlin definition. The radiological and partial pressure of oxygen, arterial (Pao2)/fraction of inspired oxygen (Fio2) criteria for ARDS had to be met within a 24-hour period, and the administration of the first dose of the study drug had to occur within 48 hours of the diagnosis of ARDS. The last patient visit was on March 6, 2018. Interventions Patients were randomized to receive an intravenous injection of 10 μg of IFN-β-1a (144 patients) or placebo (152 patients) once daily for 6 days. Main Outcomes and Measures The primary outcome was a score combining death and number of ventilator-free days at day 28 (score ranged from -1 for death to 27 if the patient was off ventilator on the first day). There were 16 secondary outcomes, including 28-day mortality, which were tested hierarchically to control type I error. Results Among 301 patients who were randomized (mean age, 58 years; 103 women [34.2%]), 296 (98.3%) completed the trial and were included in the primary analysis. At 28 days, the median composite score of death and number of ventilator-free days at day 28 was 10 days (interquartile range, -1 to 20) in the IFN-β-1a group and 8.5 days (interquartile range, 0 to 20) in the placebo group (P = .82). There was no significant difference in 28-day mortality between the IFN-β-1a vs placebo groups (26.4% vs 23.0%; difference, 3.4% [95% CI, -8.1% to 14.8%]; P = .53). Seventy-four patients (25.0%) experienced adverse events considered to be related to treatment during the study (41 patients [28.5%] in the IFN-β-1a group and 33 [21.7%] in the placebo group). Conclusions and Relevance Among adults with moderate or severe ARDS, intravenous IFN-β-1a administered for 6 days, compared with placebo, resulted in no significant difference in a composite score that included death and number of ventilator-free days over 28 days. These results do not support the use of IFN-β-1a in the management of ARDS. Trial Registration ClinicalTrials.gov Identifier: NCT02622724.

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast.