Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction

Abstract

Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.

Fig. 1

CD24 and Siglec G protect mice against inflammation and mortality associated with polybacterial sepsis. a. Targeted mutations of CD24 or Siglecg genes increased mortality. Age-matched male mice received antibiotics and CLP using 23G3/4 needles. The mice were observed twice daily for 14 days. Data shown are Kaplan Meier analysis, with statistical significance determined by log rank test. b. Targeted mutation of either CD24 or Siglecg gene increased the production of inflammatory cytokines IL-6 and TNFα. Serum samples harvested at 12 or 24 hours after CLP were measured by cytokine beads array. Data are means+/-S.D. (n=5). c-g. Targeted mutation of either the Siglecg or the CD24 gene exacerbates sepsis without increasing bacterial colony forming units (CFU) in the blood. The 21G needles were used and the CLP mice received no antibiotics. c. Survival of WT, Cd24^-/-, Siglecg^-/- mice. The X-axis shows hours after CLP, while the Y-axis shows % of live mice. Data shown are summary of five experiments, each involving 10 mice per group. d. Bacterial burdens in the blood samples (CFU/ml) harvested at 12 hours after CLP (n=8). e. Elevation of inflammatory cytokines in mice with targeted mutation of either CD24 or Siglecg at 12 hours after CLP (n=8). f. Inflammatory cytokines in the WT mice 24 hours after CLP. Data from mutant CLP mice were not collected due to mortality. g. CD24^-/- and Siglecg^-/- mice exhibit acute organ failures after CLP. Note increased alveolar and interstitial hemorrhage in lung (marked as He in top panel), massive hemorrhage and venous congestion (marked as He in renal medulla and collecting tubules (middle panels), and focal tubular necrosis with vacuolar degeneration and nuclear pyknosis and karyolysis in kidney (marked by yellow circles), at 12 hours after CLP. All data presented have been validated by 2-5 independent experiments.

Fig. 2

Expression of CD24 predominantly on dendritic cells (DC) conveys protection against sepsis. a. CD24^-/- mice that expressed CD24 under the control of CD11c promoter, CD24^-/-;CD24^Cd11ctg. Data shown are FACS profiles depicting pattern of CD24 expression in the H-2I-A^b+CD11c^- and H-2I-A^b+CD11c⁺ splenocytes of WT, CD24^-/- and CD24^-/-;CD24^Cd11ctg mice. b. Expression of CD24 on DC increased mouse survival after CLP. A 23G3/4 needle was used for puncture and no antibiotics were used. CD24^-/-;CD24^cd11ctg mice and their CD24^-/- littermates were treated by CLP and monitored for their survival. c. Transgenic expression of CD24 had no effect on blood bacterial burden at 24 hours after CLP (n=5). d. CD24 expression on DC suppressed production of inflammatory cytokines at 24 hours (n=5). The protection of DC-predominant CD24 against lethality and cytokine production has been observed in 4 independent experiments. CFU data are representative of those from two independent experiments.

Fig. 3

CD24-Siglec 10 interaction depends on sialyation of CD24. a. Biotinylated CD24Fc were pretreated with either control buffer (lane 1) or sialidase from Streptococcus pneumoniae (lane 2, specific for cleaving α2–3-linked sialic acids), Clostridium perfringens (lane 3, active for α2–6- or α2–3-linked sialosides), or Vibrio cholerae (lane 4, active for α2–3-, α2–6- or α2–8-linked sialosides) overnight at 37°C. The Siglec 10Fc fusion protein was incubated with the digested CD24Fc, and the complex was pulled down with streptavidin beads. The amounts of bead-bound Siglec 10Fc and CD24Fc were determined by Western blot with antibodies specific for either Siglec 10 or CD24. b. Efficient inhibition of CD24-Siglec 10 interaction by sialosides. Siglec 10Fc were preincubated with given concentration of either Neu5Acα2–3Lac or Neu5Acα2–6Lac and then added to plate-bound CD24Fc. The CD24-bound Siglec 10Fc were measured by biotinylated anti-Siglec 10 followed by HRP-labeled streptavidin. c. Desialylation and resialylationof CD24Fc altered its electrophoresis mobility. d. Both α2–3- and α2–6-resialylations of CD24 restore Siglec 10Fc binding. e. Sialidase treatment of WT DC increase their response to HMGB1 and HSP70. Bone marrow-derived DC from WT, CD24^-/- and Siglecg^-/- mice were treated with sialidase prior to stimulation by either HMGB1 (1μg/ml) or HSP70 (7 nM). Cytokines in the supernatants were measured by cytokine beads array. f. Desialyation of CD24 barely reduced CD24Fc binding to HMGB1. Control IgG1Fc, untreated and desialyated CD24 were co-incubated with HMGB1 (1μg/ml). Protein A beads were used to pull down Fc. The amounts of HMGB1 associated with CD24Fc were determined by immunoblot with anti-HMGB1 mAb. The data shown are representative of 2-5 independent experiments.

Fig. 4

Increased circulating sialidase activity and reduction of Siglec 10 binding of CD24 in CLP mice. a. Sialidase activity in the sera of sham-, 100 μg/mouse LPS- or CLP-treated mice. Sera were collected at 12 hours after treatment (n=5). b. Pretreatment of biotinylated CD24Fc with sera from CLP mice reduced its binding to Siglec 10Fc. Data shown are co-IP with streptavidin-conjugated beads. The top panel shows the amounts of Siglec 10Fc in the precipitates as determined by Western blot. The molecular weight shift of CD24Fc is demonstrated by Western blot using HRP-labeled streptavidin in the bottom panel. c. CLP does not affect CD24 expression in spleen cells. US, unstained; SA-PE, phycoerythorin-conjugated streptavidin. d. CLP significantly reduced spleen cell binding to Siglec 10Fc. Histograms shown on top panels are FACS profiles depicting distribution of CD24 in sham-surgery (blue line) or CLP (red line) spleen cells. The bar graphs in the bottom panels present means+/-S.D. of mean fluorescence intensities (n=3). The gates used to determine % positive cells were labeled in the upper panels. e. f. CLP reduce spleen cell binding to WT and mutant Siglec G-Fc (f) without affecting the total CD24 levels (e). The bar graphs in the bottom panels present means+/-S.D. of geo-mean fluorescence intensities or % positive cells (n=3). g. CLP alters the molecular weight distribution of CD24 in the spleen cell lysates, as determined by Western blot. h &i. CLP reduces both α2–3- and α2–6-sialyation of spleen cells (h) and CD11c⁺ cells (i). MAA: Fluorescein-Maackia amurensis Lectin I, recognizing α2–3-linked terminal sialic acid. SNA: Fluorescein-Sambucus nigra (Elderberry) Bark Lectin (SNA), recognizing α2–6-linked terminal sialic acid. All data are representative of 2–3 independent experiments.

Fig. 5

Sialidase inhibitors protect mice against sepsis. a. A mixture of two sialidase inhibitors blocks serum sialidase activity. Sera from CLP mice were mixed with given doses of inhibitors, Neu5Ac2en (AC), Neu5Gc2en (GC), or both (AC+GC) prior to the assay. The sialidase activity was measured using the Amplex Red Neuraminidase assay kit. Data shown are means+/-S.D. of triplicates. b. Sialidase inhibitors prolong survival of WT but not CD24^-/- and Siglecg^-/- mice after CLP (n=10). The mice received a mixture of AC and GC (100 μg/mouse/injection) immediately prior to CLP and every 12 hours thereafter. c and d. Sialidase inhibitors had no effect on the serum bacterial CFU at 12 hours (c, left panel) or 24 hours (c, right panel) after CLP. Bacterial burden in mutant mice 24 hours after CLP were not assayed as mouse deaths biases sampling. d. Sialidase inhibitors reduce the levels of multiple inflammatory cytokines. Sera were collected at 24 hours after CLP to measure cytokines. Data shown are means+/-S.D. (n=8). e. Sialidase inhibitor protects CLP mice in combination with antibiotics. Data shown are representative of 2–5 independent experiments.

Fig. 6

S. pneumoniae sialidases exacerbate sepsis by CD24-and Sigelec G-dependent mechanism. a. Characterization of the sialidase activity of WT (D39) and nanA^-nanB^- mutant (mD39) strains. b. Serum sialidase activity of mice at 24 hours after intraperitoneal infections with 10⁴ CFU of either D39 or mD39. The sialidase activity in a and b were measured using Amplex® Red Neuraminidase Assay Kit. c. Bacterial sialidase reduces Siglec 10Fc binding to spleen cells. Representative FACS profiles are shown in the top panels, while the summary data were shown at the lower panel (n=3). d. Bacterial sialidases exacerbate sepsis in WT but not mutant mice. Data shown were Kaplan Meier survival analysis of accumulating data from two independent experiments (n=10), the log-rank tests were used to calculate the P value. e. Bacterial sialidases increased production of inflammatory cytokines in WT but not mutant mice. f. Deletion of gene encoding both NanA and NanB sialidases does not reduce blood bacterial burden. Data shown represents 2–3 independent experiments. N=5 unless otherwise specified.