Sema7A is crucial for resolution of severe inflammation

Abstract

Endogenous mediators regulating acute inflammatory responses in both the induction and resolution phases of inflammatory processes are pivotal in host defense and tissue homeostasis. Recent studies have identified neuronal guidance proteins characterized in axonal development that display immunomodulatory functions. Here, we identify the neuroimmune guidance cue Semaphorin 7A (Sema7A), which appears to link macrophage (MΦ) metabolic remodeling to inflammation resolution. Sema7A orchestrated MΦ chemotaxis and chemokinesis, activated MΦ differentiation and polarization toward the proresolving M2 phenotype, and promoted leukocyte clearance. Peritoneal MΦ^Sema7A-/- displayed metabolic reprogramming, characterized by reductions in fatty acid oxidation and oxidative phosphorylation, increases in glycolysis and the pentose phosphate pathway, and truncation of the tricarboxylic acid cycle, which resulted in increased levels of the intermediates succinate and fumarate. The low accumulation of citrate in MΦ^Sema7A-/- correlated with the decreased synthesis of prostaglandins, leading to a reduced impact on lipid-mediator class switching and the generation of specialized pro resolving lipid mediators. Signaling network analysis indicated that Sema7A induced the metabolic reprogramming of MΦ by activating the mTOR- and AKT2-signaling pathways. Administration of Sema7A^SL4cd orchestrated the resolution response to tissue homeostasis by shortening the resolution interval, promoting tissue protection in murine peritonitis, and enhancing survival in polymicrobial sepsis.

Keywords: Semaphorin 7A; inflammation; lipid mediator; metabolism; resolution.

Fig. 1.

Sema7A controls the macrophage inflammatory phenotype and regulates human macrophage chemotaxis and chemokinesis. Human PBMCs were stimulated with GM-CSF, M-CSF, or Sema7A^SL4cd for 7 d, and Sema7A transcript expression in differentiated M1 and M2 MΦs was quantified by RT-PCR (n = 14 to 16) (A). (B) Cell morphology was analyzed by phase contrast images and measurements of the cell shape, length, and perimeter (magnification 200×). (Scale bar: 20 μm.) n = 210. (C) The expression levels of key genes that contribute to M2 differentiation, Arg1 and CD163, and central genes of M1 differentiation, STAT-1 and CD80, were analyzed (n = 11). (D and E) Phenotypic polarization of macrophages: M1 MΦs were sequentially challenged with Sema7A^SL4cd and TNF-α or vehicle for 24 h. The gene expression levels of M1 polarization markers (including STAT-1, CD40, CD80, IL-1β, and IL-6), key genes of M2 polarization (such as Arg1, CD163, CD206, and IL-10) and the ALX/FPR2 and GPR32 receptors were quantified by RT-PCR (n = 13 to 20). (F) Schematic model of the microfluidic migration chamber. A chemoattractive gradient with a monocyte chemotactic protein (MCP-1) and Sema7A^SL4cd was established between eight peripheral wells and a central cell loading well. M1 MΦ chemotaxis was evaluated using a Casy TT cell counter (Omni Life Science) (n = 10). (G) The rate of MΦ clearance of fluorescence-labeled ZyA particles and human apoptotic PMNs was assessed photometrically (n = 5 to 9). (H and I) MΦs were transfected with integrin α₁-, integrin αv-, or integrin β₁-siRNA or treated with an anti-Plexin C1 and then stimulated with Sema7A^SL4cd antibody. MΦ clearance of fluorescence-labeled ZyA particles was then assessed photometrically (n = 4 to 10). (J) The colocalization of integrin heterodimers and integrins with Sema7A was assessed with the Similarity feature of the ImageStreamx mkII system. The results are representative of three to eight independent experiments and are expressed as the mean ± SEM; significance was determined by one-way ANOVA with Bonferroni correction (B–I) or the unpaired two-tailed Student’s t test (A). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Sema7A drives cellular metabolism. Glycolytic capacity and mitochondrial respiration were assessed by using the Seahorse Glycolysis and Mito Stress tests. MФs^SEMA7A+/+ and MФs^{SEMA7A−/−} were subjected to peritoneal lavage, and ECARs were measured after consecutive injections of glucose, oligomycin, and 2-DG. Oxygen consumption rates were determined after injections of oligomycin, FCCP, rotenone, and antimycin A. Glycolytic and mitochondrial respiration markers were calculated (A) at baseline and (B) following stimulation with ZyA for 4 h (n = 20). The glucose metabolism pathways in MФs^SEMA7A+/+ and MФs^{SEMA7A−/−} were assessed via the PCR-based analysis of enzymes involved in glucose metabolism. Intracellular metabolites were quantified by NMR spectroscopy (n = 3). The experiment was conducted (C–F) at baseline and (G) following stimulation with ZyA for 4 h. (H) ECAR and OCR were also determined in MФs from C57/BL/6 mice that were stimulated either with vehicle (MФ^vehicle) or the peptide Sema7A^SL4cd (MФ^{SEMA7A−SL4cd}) followed by ZyA stimulation for 0 and 4 h. Samples were pooled from three to four mice in each group. The results represent three independent experiments and are expressed as the mean ± SEM; significance was determined by the unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.

Fig. 3.

Sema7A-dependent macrophage intracellular signaling. SEMA7A^+/+ and SEMA7A^−/− animals were injected intraperitoneally with ZyA, and peritoneal MΦs were collected 4 h later. (A) The mTOR- and AKT-signaling pathways were assessed by protein expression and phosphorylation analysis by using a protein microarray. Samples were pooled from three to four mice in each group for each experiment. (B) Schematic model of the cellular effects of Sema7A.

Fig. 4.

SEMA7A^−/− mice display deficient inflammation resolution. Sema7A-deficient mice and littermate controls were injected with ZyA, and peritoneal lavages were collected at 4, 12, 24, and 48 h. (A) Total leukocytes were counted by light microscopy, and PMNs were identified by flow cytometry (n = 6 to 16). Resolution indices. (B) The cytokine levels of IL-1β, IL-6, and KC in peritoneal fluids were measured by enzyme-linked immunosorbent assay (ELISA) (n = 5 to 6). (C) Classical and nonclassical monocytes and MΦs as well as monocyte-derived MΦ efferocytosis were quantified by flow cytometry (n = 6 to 16). (D) The levels of bioactive lipid mediators and precursors derived from arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) in the peritoneal fluids of SEMA7A^+/+ and SEMA7A^−/− animals treated with ZyA (n = 15 to 19) for 4 h were quantified by LC-MS/MS–based profiling. (E) PCNA expression in peritoneal slices (24 h after ZyA injection) as detected by immunohistochemistry (n = 4) and the calculated indices (40× magnification). (Scale bar: 50 µm.) (F) Survival rates and normalized body temperatures of SEMA7A^+/+ and SEMA7A^−/− animals that underwent the CLP procedure with median survival and hazard ratios (WT: n = 9; KO: n =13). The results represent at least two independent experiments and are expressed as the mean ± SEM (A–E) and the geometric mean (F); significance was determined by the unpaired two-tailed Student’s t test (A–E) or log-rank test (F). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Exogenous Sema7A activates resolution programs. WT animals were sequentially injected with ZyA and vehicle or Sema7A^SL4cd, and lavages were collected at 4, 12, 24, and 48 h. (A) Total leukocytes were counted by light microscopy, and PMNs and classical and nonclassical monocytes and (B) MΦ efferocytosis were assessed by flow cytometry (n = 6 to 17). (C) Resolution indices. (D) The levels of bioactive lipid mediators and precursors, including those in the AA, DHA, and EPA pathways, in the peritoneal fluids of WT animals that were treated with ZyA and Sema7A^SL4cd or vehicle for 4 or 12 h (n = 9 to 19) were quantified by LC-MS/MS–based profiling. (E) WT animals were injected with ZyA and treated with vehicle or Sema7A^SL4cd after 4 h at the peak of inflammation, and lavages were collected at 12, 24 and 48 h. Classical and nonclassical monocytes and MΦs as well as monocyte-derived macrophage efferocytosis were quantified by flow cytometry (n = 8 to 13). Resolution indices were determined. (F) The survival rates of WT animals treated daily with Sema7A^SL4cd or vehicle that underwent the CLP procedure and the median survival (n = 9 to 10). The results represent three independent experiments and are expressed as the mean ± SEM (A–E); significance was determined by the unpaired two-tailed Student’s t test (A, B, D, and E), and the log-rank test (F), *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Sema7A in pediatric ICU patients with abdominal compartment syndrome. Plasma samples from 143 children in the ICU with and without ACS were collected within 24 h after admission and on the day of discharge from the PICU of Hannover Medical School. (A) Representative images displaying critically ill children with diaphragmatic elevation (Left), abdominal diastolic bloodflow in a patient with IAH (Middle) and a patient with ACS (after decompressive laparotomy with the establishment of an open abdomen/laparostoma to reduce intra-abdominal pressure and the associated tissue and organ impairments) (Right). (B) Sema7A levels were measured by ELISA on the day of admission to the ICU and on the day of discharge. (C) Overview of ICU patient characteristics with a PRISM score ≤13 and a PRISM score >13. (D and E) Correlation between Sema7A and the clinical parameters of patients in the PICU with PRISM III scores ≤13 (D) and >13 (E) on the day of admission. Spearman’s rank correlation coefficient Rho and the corresponding 95% CI interval are shown. The results are displayed as the mean ± SEM. CI; significance was determined by the nonparametric Mann–Whitney U test (B); correlation was assessed using the Spearman’s rank correlation test (D and E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.