SLAMF7 regulates the inflammatory response in macrophages during polymicrobial sepsis

Uncontrolled inflammation occurred in sepsis results in multiple organ injuries and shock, which contributes to the death of patients with sepsis. However, the regulatory mechanisms that restrict excessive inflammation are still elusive. Here, we identified an Ig-like receptor called signaling lymphocyte activation molecular family 7 (SLAMF7) as a key suppressor of inflammation during sepsis. We found that the expression of SLAMF7 on monocytes/macrophages was significantly elevated in patients with sepsis and in septic mice. SLAMF7 attenuated TLR-dependent MAPK and NF-κB signaling activation in macrophages by cooperating with Src homology 2–containing inositol-5′‑phosphatase 1 (SHIP1). Furthermore, SLAMF7 interacted with SHIP1 and TNF receptor–associated factor 6 (TRAF6) to inhibit K63 ubiquitination of TRAF6. In addition, we found that tyrosine phosphorylation sites within the intracellular domain of SLAMF7 and the phosphatase domain of SHIP1 were indispensable for the interaction between SLAMF7, SHIP1, and TRAF6 and SLAMF7-mediated modulation of cytokine production. Finally, we demonstrated that SLAMF7 protected against lethal sepsis and endotoxemia by downregulating macrophage proinflammatory cytokines and suppressing inflammation-induced organ damage. Taken together, our findings reveal a negative regulatory role of SLAMF7 in polymicrobial sepsis, thus providing sights into the treatment of sepsis.

Escherichia coli (E.coli, ATCC 25922) was grown in LB agar (Difco, BD). Bacterial in logarithmic growth were collected, counted on agar plates and then resuspended in 0.9% NaCl for following infected experiments.

BMDM and human monocytes isolation
Murine BMDMs were obtained as previously described (1). Briefly cells were maintained in DMEM supplemented with 10% FBS and 10% L929 cell supernatant as conditioned medium providing macrophage colony stimulating factor. BMDMs were obtained as a homogeneous population of adherent cells after 7 days culture. The purity of the differentiation method was > 95% as a routinely confirmed by flow cytometry. Human monocytes were isolated from peripheral venous blood from healthy subjects. Monocytes were isolated from the peripheral blood mononuclear cells (PBMCs) layer according to Ficoll manufacturers (TBD, Tianjin, China). An ammonium chloride-based lysis (BD Biosciences) was performed to remove erythrocytes. PBMCs were then stained in PBS containing 1% BSA with monoclonal antibodies to CD14 (BD Biosciences) for sorting. Purified monocytes were cultured at 1x10 6 cells/ml overnight before use.

Retrovirus-mediated gene transduction
For making RAW264.7 stable cell lines, the cDNA encoding full-length mouse SLAMF7 (NCBI Gene ID: 75345) was cloned into pSin-EF2-puro-oligo transfer plasmid. For deletion of SLAMF7 gene in RAW264.7, sgRNAs were cloned into pXPR_001 plasmid according to Zhang's lab lentiCRISPR system (2). Retrovirus expressing the appropriate alleles was produced by 293FT cells co-transfected with the packaging system composed of psPAX2 packaging plasmid and pMD2.G  Table 4.

ELISA assay
Cell culture medium, mouse serum, peritoneal lavage and organ grinding fluid were collected and cytokines production including TNF-, IL-1 and IL-6 were measured by murine ELISA Kit (R&D System, Minncapolis, MN) according to the manufacturers' instructions.

Colony forming unit (CFU) assay
Peritoneal lavage fluid (PLF) and blood for bacterial culture were collected as mice were euthanized at 18 hours after CLP. PLF and blood were subjected to serial 10-fold dilutions and cultured at 37℃ overnight in 5% sheep blood agar (Teknova). CFUs were quantified by manual counting.

Phagocytosis Assay Assessed by Flow Cytometry
Phagocytosis measured by flow cytometry was performed as described previously (3). Adherent macrophages were then co-cultured with Filmtracer Green Biofilm (FTGB, Invitrogen)-labeled PA or E.coli for 1 hour. Following 1 hour bacterial incubation, cells were washed three times utilizing cold PBS and centrifuged to remove the extracellular bacteria. The. The mean fluorescence intensity (MFI) was calculated with flowjo software and used as a measure of phagocytosis.

Confocal microscopy and histological staining
Cells were grown on Glass Bottom Cell Culture Dish were fixed with 4% paraformaldehyde (BBI Life Science, Shanghai, China) followed by membrane permeabilization, blocking and antibody incubation. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) and mounted with ProLong Gold antifade reagent from Invitrogen and viewed by confocal microscopy (Zeiss, LSM780, German). For histological section staining, mouse lung tissue samples were fixed in 4% PFA for 24 h and embedded in paraffin. Four-μm-thick sections were deparaffinized and stained with hematoxylin and eosin to determine organ damage and leukocyte infiltration. To calculate the lung injury scores from H&E staining, 5 random high-power fields were independently scored in a blinded fashion for each slice. The features of lung injury, including neutrophils in the alveolar space, neutrophils in the interstitial space, hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening, were determined and weighted according to the relevance ascribed to each feature and then were normalized to the number of fields evaluated (4).