TLR4-Dependent Secretion by Hepatic Stellate Cells of the Neutrophil-Chemoattractant CXCL1 Mediates Liver Response to Gut Microbiota

Background & Aims The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. Methods Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. Results TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. Conclusions Showing the specific activation of TLR4 and the secretion of one major functional chemokine— CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state.National Institute of Allergy and Infectious Diseases (U.S.) (Grant #1R01AI072049

Malaria Sporozoites Traverse Host Cells within Transient Vacuoles

International audiencePlasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication

Decrease of CXCL1 message and neutrophil counts in TLR4 deficient liver and after antibiotic treatment.

<p><b>(A)</b> CXCL1 expression in the total liver as analyzed by microarrays. Mean values were obtained from three Genechips for three WT and three TLR4 deficient mice. Statistically significant differences between WT and TLR4 deficient mice are indicated by an asterisk, *<i>P</i><.05, Student <i>t</i> test. (<b>B)</b> CXCL1 expression measured by quantitative RT-PCR. The relative quantity of CXCL1 mRNA in the total liver of WT and TLR4 deficient mice is indicated (*<i>P</i><.01). <b>(C)</b> Relative expression of CXCL1 in the liver from untreated or antibiotic-treated (ABT) WT mice and TLR4 deficient mice; *<i>P</i><.01 <b>(D)</b> Neutrophils counts in the total liver. CD11+ Gr1^high TCR- cells among total live leukocytes isolated from WT and TLR4 deficient liver. In Fig 1B, 1C and 1D, data are representative of five separate experiments with six WT mice (treated or not with antibiotics) and five TLR4 mice; ^<b>#</b><i>P</i><.05; unpaired Mann -Whitney test.</p

Neutrophils migrate in response to CXCL1 secretion following TLR4 activation in hepatic stellate cells.

<p><b>(A)</b> Schematic representation of the neutrophil chemotaxis assay. <b>(B)</b> Quantification of neutrophil migration in response to secretory WT or TLR4 deficient stellate cells. WT stellate cells were treated (WT HSC + anti CXCL1) or not with anti-CXCL1 antibody. As for internal positive control, the migration of neutrophils towards TLR4 deficient stellate cells supplemented with CXCL1 protein (TLR4 HSC + CXCL1) and with CXCL1 protein only (CXCL1) was quantified in only one experiment. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between WT HSCs and TLR4 deficient HSCs, as well as between WT HSCs treated or not with anti-CXCL1, are indicated.</p

Hepatic stellate cells are the major source of CXCL1, as shown by both quantification of secretion and <i>in situ</i> localization.

<p><b>(A)</b> Quantification of CXCL1 secretion in enriched fractions of hepatocytes, KCs, LSECs and HSCs, freshly isolated and stimulated <i>in vitro</i> with LPS (1 ng/mL LPS, black squares) during 24 hours. Data are representative of three separate experiments with six mice in each group; ^<b>#</b><i>P</i><.05. <b>(B)</b> <i>In-situ</i> localization of CXCL1 in the liver. Immunofluorescent detection for CXCL1 (red) and liver cells nuclei (blue) for nuclei first shows CXCL1 expression in the sinusoids throughout liver parenchyma. <b>(C)</b> Higher resolution shows that CXCL1 (red) is expressed by sub-endothelial cells, which also store retinol droplets in separate compartments, as shown by CRBP1 staining (green). The Cellular Retinol Binding Protein-1 (CRBP-1) is the best marker to detect simultaneously both resting (Glial Fibrillary Acidic Protein, GFAP+) and activated (α-Smooth Muscle Actin, αSMA+) stellate cells <i>in situ</i>. Alexa Fluor-546-CXCL1 (red) staining does not colocalize either with Tie2-GFP in LSECs (green, <i>upper panel</i>), or F4/80 in KCs (blue, <i>middle panel</i>), but with AlexaFluor-488-CRBP1 (green, <i>lower panel</i>), staining both resting and activated HSCs. TOPRO3 was used for nuclei vizualisation.</p

Cytokine secretion by hepatocytes, KCs, LSECs and HSCs after isolation from the same liver and in response to low levels of LPS.

<p>Liver cells were freshly isolated on density gradient followed by cell sorting and stimulated with LPS (1ng/mL LPS, black bars or 100ng/mL LPS, hatched bars). Cytokine secretion was measured in the same supernatant with a multiplex assay, run in triplicates. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between basal LPS stimulation (1ng/mL) and higher LPS stimulation (100ng/mL) are indicated. Lower panel: bright field images of cells right after isolation (Hepatocytes, LSECs, KCs). Images of HSCs at higher resolution show the retinol droplets at Day 0 and the typical shape of the activated stellate cells after 4 days in culture.</p

Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.

<p>Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.</p

High serum LDH and liver metastases are the dominant predictors of primary cancer resistance to anti-PD(L)1 immunotherapy

International audienceAim: Anti-PD-(L)1 immunotherapies improve survival in multiple cancers but remain ineffective for most patients. We applied machine-learning algorithms and multivariate analyses on baseline medical data to estimate their relative impact on overall survival (OS) upon anti-PD-(L)1 monotherapies. Method: This prognostic/predictive study retrospectively analysed 33 baseline routine medical variables derived from computed tomography (CT) images, clinical and biological meta-data. 695 patients with a diagnosis of advanced cancer were treated in prospective clinical trials in a single tertiary cancer centre in 3 cohorts including systemic anti-PD-(L)1 (251, 235 patients) versus other systemic therapies (209 patients). A random forest model combined variables to identify the combination (signature) which best estimated OS in patients treated with immunotherapy. The performance for estimating OS [95%CI] was measured using Kaplan–Meier Analysis and Log–Rank test. Results: Elevated serum lactate dehydrogenase (LDHhi) and presence of liver metastases (LM+) were dominant and independent predictors of short OS in independent cohorts of melanoma and non-melanoma solid tumours. Overall, LDHhiLM+ patients treated with anti-PD-(L)1 monotherapy had a poorer outcome (median OS: 3.1[2.4–7.8] months]) compared to LDHlowLM-patients (median OS: 15.3[8.9-NA] months; P &lt; 0.0001). The OS of LDHlowLM-patients treated with immunotherapy was 28.8[17.9-NA] months (vs 13.1[10.8–18.5], P = 0.02) in the overall population and 30.3[19.93-NA] months (vs 14.1[8.69-NA], P = 0.0013) in patients with melanoma. Conclusion: LDHhiLM+ status identifies patients who shall not benefit from anti-PD-(L)1 monotherapy. It could be used in clinical trials to stratify patients and eventually address this specific medical need

TTC7A mutations disrupt intestinal epithelial apicobasal polarity

Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain–7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development