Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza

Abstract

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Fig. 1

Slit2N stabilizes the endothelium in vitro by enhancing VE-cadherin localization at the cell surface. (A) In vitro permeability was measured in HMVEC-lung cells stimulated with LPS, TNF-α, or IL-1β in the presence of Mock (see Materials and Methods) or Slit2N. (B) Robo4 or control siRNA knockdown-treated HMVEC-lung cells were stimulated with IL-1β in the presence of Mock or Slit2N to assess permeability in vitro. (C to E) HMVEC-lung cells were treated with Mock or Slit2N and subjected to membrane fractionation and subsequent immunoblotting for VE-cadherin (C), p120-catenin (D), or β-catenin (E). (F) HMVEC-lung cells were stimulated with Mock or Slit2N and subjected to immunofluorescence for VE-cadherin (green). White arrows, areas of enhanced VE-cadherin cell surface localization. For all experiments, n ≥ 3, and error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Fig. 2

Slit2N enhances a VE-cadherin–p120-catenin interaction in vitro. (A) HMVEC-lung cells were stimulated with IL-1β in the presence of Mock or Slit2N and immunostained for VE-cadherin and p120-catenin. White arrows, cell surface areas lacking VE-cadherin or p120-catenin in Mock-treated cells; yellow arrows, areas of enhanced cell surface localization of VE-cadherin or p120-catenin in Slit2N-treated cells. (B) HMVEC-lung cells were stimulated with IL-1β in the presence of Mock or Slit2N. Lysates were subjected to immunoprecipitation for VE-cadherin followed by immunoblot for p120-catenin and VE-cadherin. (C) HMVEC-lung cells were labeled with an antibody to VE-cadherin and stimulated with IL-1β in the presence of Mock or Slit2N. Cells were acid-washed to strip the surface-bound VE-cadherin (top row). VE-cadherin internalization (green) was assessed. White arrows, areas of internalization. (D) In vitro permeability was measured in the presence of a control IgG or VE-cadherin antibody (Ab). For all experiments, n ≥ 3, and error bars represent SEM. **P < 0.01.

Fig. 3

Slit2N inhibits LPS-induced permeability, protein exudates, and cell infiltrates in vivo. (A) Robo4^+/+ and Robo4^AP/AP mice were given an intravenous injection of Mock or Slit2N followed by intratracheal instillation of LPS (10 µg). Mice later received an intravenous injection of EBA, and EBA accumulation in the lungs was used to assess vascular permeability (n ≥ 4). (B to D) Twenty-four hours after LPS administration, bronchoalveolar lavages were obtained and assessed for protein content (B), total inflammatory cell accumulation (C), or neutrophil accumulation (saline value is too low to be visible) (D) (n ≥ 5). (E) H&E staining was performed on lung sections from mice exposed to LPS in the presence of Mock or Slit2N. (F) Protein exudates measured in mice treated with 3.3 µg of LPS (n = 5). (G to I) Mice were given an intravenous injection of Mock or Slit2N with control or VE-cadherin–blocking antibody followed by intratracheal instillation of LPS. Bronchoalveolar lavages were obtained and assessed for protein content (G), total inflammatory cell accumulation (H), or neutrophil accumulation (I) (n ≥ 5). Error bars represent SEM. *P < 0.05, **P < 0.01, ****P < 0.001.

Fig. 4

Slit2N reduces permeability and mortality in a CLP model of sepsis. (A and B) Mice were subjected to CLP or sham operation and then given an intravenous injection of EBA. EBA accumulation was measured in the kidney (A) or spleen (B) to assess vascular permeability (n = 5). (C) Robo4^+/+ mice were subjected to CLP and treated with Mock or Slit2N, and survival was assessed (Mock-treated, n = 15; Slit2N-treated, n = 14). (D and E) Cytokine (D) and chemokine (E) concentrations in the serum of Mock- or Slit2N-treated CLP mice (n = 6). (F) Robo4^AP/AP mice were subjected to CLP and treated with Mock or Slit2N, and survival was assessed (Mock-treated, n = 13; Slit2N-treated, n = 13). Error bars represent SEM. *P < 0.05, **P < 0.01, ****P < 0.001.

Fig. 5

Slit2N reduces mortality in models of H5N1 infection. (A) BALB/c mice were infected intranasally with H5N1 virus. Mice were given an intravenous injection of EBA, and EBA accumulation was measured in the lungs to assess vascular permeability (n = 5). (B) Mouse survival after H5N1 infection (Mock-treated, n = 20; Slit2N-treated, n = 20). (C) H&E staining was performed on lung sections from H5N1-infected mice 6 days after infection. White arrows in the top left panel show accumulation of edema fluid around a pulmonary arteriole. The top middle panel demonstrates exuberant alveolar inflammation. The black arrow in the top right panel shows the presence of foamy macrophages. (D) H5N1 viral titers were measured 6 days after infection (n = 3 groups of pooled mice). (E and F) Cytokine (E) or chemokine (F) concentrations measured in lung homogenates 6 days after infection (n = 3 groups of pooled mice). *P < 0.05. NS, not significant.

Fig. 6

Slit reduces vascular leak caused by multiple inflammatory stimuli through enhancing VE-cadherin at the cell surface. (A) Under normal conditions, alveolar capillaries are semipermeable. (B) Inflammatory stimuli cause a large release of cytokines, leading to internalization of VE-cadherin and disruption of barrier function. This results in vascular leak and accumulation of protein-rich edema fluid in the alveolar space. (C) Slit enhances vascular barrier function against multiple cytokines by enhancing VE-cadherin at the cell surface.