PD-1 blockade improves Kupffer cell bacterial clearance in acute liver injury

Patients with acute liver failure (ALF) have systemic innate immune suppression and increased susceptibility to infections. Programmed cell death 1 (PD-1) expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of the programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) pathway in regulating Kupffer cell (KC) inflammatory and antimicrobial responses in acetaminophen-induced (APAP-induced) acute liver injury. Using intravital imaging and flow cytometry, we found impaired KC bacterial clearance and systemic bacterial dissemination in mice with liver injury. We detected increased PD-1 and PD-L1 expression in KCs and lymphocyte subsets, respectively, during injury resolution. Gene expression profiling of PD-1+ KCs revealed an immune-suppressive profile and reduced pathogen responses. Compared with WT mice, PD-1–deficient mice and anti–PD-1–treated mice with liver injury showed improved KC bacterial clearance, a reduced tissue bacterial load, and protection from sepsis. Blood samples from patients with ALF revealed enhanced PD-1 and PD-L1 expression by monocytes and lymphocytes, respectively, and that soluble PD-L1 plasma levels could predict outcomes and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for the PD-1/PD-L1 axis in suppressing KC and monocyte antimicrobial responses after liver injury and identifies anti–PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.


A. Supplemental Figures, Tables and Videos
Video S1. Intravital imaging of Kupffer cell bacterial capture in the liver.
Video S2. 3D reconstitution of liver intravital imaging with increasing transparency showing bacteria inside Kupffer cells. Table S1: Clinical and physiological parameters of patients with acute liver failure (ALF) -soluble PD-L1 levels. Table S2. Primers used for quantitative reverse transcription PCR (RT-qPCR) in WT and PD-1 -/mice. Table S3. Anti-mouse monoclonal antibodies used for phenotypic characterization of mouse blood immune cells. Table S4. Anti-mouse monoclonal antibodies used for phenotypic characterization of mouse liver myeloid cells. Table S5. Anti-mouse monoclonal antibodies used for phenotypic characterization of mouse liver lymphoid cells. Table S6. Liver tissue weights of WT and PD-1 -/mice used for flow cytometry experiments. Table S7. Liver tissue weights of WT and PD-1 -/mice used for bacterial burden (CFU) experiments. Table S8. Anti-human monoclonal antibodies used for phenotypic characterization of human blood monocytes. Table S9. Anti-human monoclonal antibodies used for phenotypic characterization of human blood.

Video S2. 3D reconstitution of liver intravital imaging with increasing transparency showing bacteria inside
Kupffer cells. Liver intravital imaging was performed in WT mice intravenously challenged with GFP-expressing E. coli. Time-lapse video of a mouse at steady-state conditions shows captured E. coli (Green, GFP, pseudocolor) by Kupffer cells (Purple, anti-F4/80 labelled, pseudocolor). 3D reconstitution with increasing transparency of Kupffer cells indicated that the trapped E. coli were inside Kupffer cells. Scale bars: 50 μm.

Quantitative reverse transcription PCR (RT-qPCR)
Liver tissue was collected and snap frozen from baseline (control) and APAP-treated mice.
Tissues were disrupted in TissueLyser II (Qiagen) and total RNA was extracted by RNeasy Plus Mini Kit (Qiagen). RNA quality and quantity were assessed by OD reading at 260nm and 280nm. One microgram (1 μg) of total RNA was converted to cDNA using SuperScript IV reverse transcriptase with Random Hexamers (Invitrogen). Targeted and housekeeping genes were identified using Ensembl database (Mouse (GRCm38.p5) and RefSeq accession number used to design qPCR primers using NCBI/Primer-BLAST (Suppl.

Isolation of liver leukocytes
Isolation of mouse liver non-parenchymal cells was performed. Livers were harvested from PBS-perfused mice, chopped finely and incubated for 45 min with digestion buffer [5% fetal bovine serum (Gibco), 0.5 mg/mL Collagenase VIII from Clostridium histolyticum (Sigma-Aldrich, UK), 0.1 mg/mL Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, UK) in Dulbecco's PBS with calcium and magnesium (Gibco)] in a 15 mL. Falcon tubes on a shakerincubator at 250 rpm (37 o C, 5% CO2). After incubation, single cell suspensions were filtered over a 100 μm filter (BD Biosciences) attached to 50 mL. Falcon tubes. The samples were next subjected to two cycles of washing with Dulbecco's PBS without calcium and magnesium (Gibco) at 400 rpm, 4°C for 5 min from which the supernatant was kept, omitting the parenchymal cell pellet. Subsequently, the supernatant was centrifuged at 1.400 rpm, 4°C for 5 min and the cell pellet was lysed for blood erythrocytes by 2 min room temperature incubation with ACK lysing buffer (Gibco) followed by a was with PBS.

Flow cytometry of blood immune cells
Circulating blood samples were taken by cardiac puncture and collected in blood collection tubes (Microvette, Sarstedt) to prevent clotting. Blood samples (100 μL) were added into FACS tubes and lysed in 2 mL RBC Lysis Buffer (1X) (Invitrogen) for 10 min at room temperature in the dark. After incubation, FACS tubes were spun down (5 min, 500g at room temperature) and cells were resuspended in 100 μL FACS buffer (PBS with 2% FBS). All samples were preincubated for 10 min at room temperature with TruStain fcX TM (anti-mouse CD16/32) antibody (BioLegend) and then stained for 25 min at room temperature in the dark with monoclonal antibodies detailed in (Suppl.

Flow cytometry of liver immune cells
Liver leukocytes were isolated as described above. Following ACK lysing buffer (Gibco) incubation and PBS wash, cells were transferred into FACS tubes, resuspended in 100 μL FACS buffer (PBS with 2% FBS). All samples were pre-incubated 10 min at room temperature with TruStain fcX TM (anti-mouse CD16/32) antibody (BioLegend) prior to staining for 25 min (room temperature, in the dark) with monoclonal antibodies detailed in (Suppl.
Supplemental Table 6. Liver tissue weights of wild-type (WT) and PD-1-deficient (PD-1 KO) mice used for characterization of myeloid and lymphoid cell subsets by flow cytometry before (control) and after APAP-induced acute liver injury. Liver tissue sampling for flow cytometry experiments was performed as described above (page 18).