Pandemic influenza and bacterial sepsis are infections that have sizable mortality rates and substantially affect public health (32
). New ways to address these constantly evolving biological threats are needed (33
). Here, using rodent models of infection, we have demonstrated that cytokine storm and capillary leakage contribute to the poor outcome of endotoxin-induced acute lung injury, polymicrobial sepsis, and pandemic influenza. We further show that administration of the exogenous ligand Slit2N strengthens the endothelial barrier and blunts vascular leak in response to cytokine storm (). Exogenously applied Slit2N stabilizes cell surface VE-cadherin, a primary molecular determinant of intact barrier function in the endothelium, and this strengthened barrier can protect mice from the lethal effects of sepsis or influenza infection. Furthermore, in genetically modified mice lacking Robo4, Slit2N cannot prevent vascular leakage in infection or sepsis, indicating that Robo4 is a critical mediator of Slit-promoted vascular stability.
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 (more ...)
We and others have long assumed that Robo4 played an essential role in development based on a number of observations, including the role of Slit-Robo signaling in neuronal guidance (34
) and the lethality of mutations in genes of this signaling pathway (35
). Yet, unexpectedly, mice homozygous for null mutations of Robo4
are viable (15
). Similarly, in a detailed characterization of lung development, we could find no structural differences between Robo4AP/AP
(null) mice and their wild-type sibling controls (fig. S7
and fig. S8
Our results suggest an alternative function for Robo4: This receptor may be important for modulating the vascular response to inflammatory cytokines but not for development. As with other pathways that maintain adult homeostasis, including many involved in the immune response, one cannot a priori expect that these pathways are also essential for development. Genetic alterations in these pathways might only manifest after exposure to physiologic or environmental stress (38
In our experiments, we tested one way that signaling via Robo4 could modulate critical host inflammatory responses. Because Slit2 affects migration of dimethyl sulfoxide (DMSO)–treated HL-60 cells, a promyelocytic leukemia cell line often used as a surrogate for primary neutrophils (39
), we asked whether Slit2 inhibits migration of neutrophils, which could account for the reduced numbers of inflammatory cells in the lungs of LPS-treated mice (40
). Although in our hands Slit2 also inhibited migration of DMSO-treated HL-60 cells, we found no evidence that primary hPMNs respond to Slit2N, nor do they express Robo receptors (fig. S3, A and B
Rather, our data demonstrate that Robo4 is required for the effect of Slit2N on VE-cadherin–mediated vascular barrier function. Similarly, expression of either Robo4 or Robo1 is necessary and sufficient to make cells sensitive to Slit (15
). The simplest interpretation of these data would be that Slit2 acts as a direct ligand for Robo4. This may not be the case, however. Others have postulated that co-receptors such as syndecans are required for the function of Robo receptors in neural guidance (42
). Western blot and immunoprecipitation studies in the presence of non-denaturing detergents (0.5% NP-40) reveal specific binding of Slit2 to Robo4 (18
), but the use of harsher conditions using ionic denaturing detergents (1% Triton X-100–0.5% deoxy-cholate) fails to preserve this interaction (46
). To reconcile this variable and detergent-dependent binding between Slit2 and cell surface Robo4, the absence of strong interaction between Slit and Robo4 in an in vitro Biacore assay (46
), and the strong signaling and functional response of Robo4 to Slit, Sheldon et al
) and Suchting et al
) propose that Robo4 and Robo1 receptors form a heterodimeric complex in human vein endothelial cells and show that the Robo1 and Robo4 receptors bind to one another. Consistent with their model, we find that knockdown of either Robo4 or Robo1 abrogates the ability of Slit to inhibit migration of human vein endothelial cells (fig. S9
). Further investigation of the roles of Robo1 and syndecans as co-receptors for Robo4 is needed.
The endothelial cell monolayer provides a critical semipermeable barrier between the blood and tissue that regulates the passage of nutrients, fluid, and leukocytes into the interstitial space (47
). The integrity of this barrier is determined by homophilic interactions between the cell surface adherens junction protein VE-cadherin on adjacent endothelial cells (48
). In states of active angiogenesis or acute inflammation, cytokines induce rapid endocytosis of VE-cadherin, disrupting the transcellular homophilic binding of VE-cadherins, deconstructing paracellular adherens junctions, and resulting in hyperpermeability (21
). Our study suggests that the destabilizing effects of angiogenic and inflammatory cytokines are opposed by extracellular cues that promote vascular stability. Although two stabilizing regulators of the vascular barrier, Tie2-dependent angiopoietin signaling and Robo4-dependent Slit signaling, use different immediate downstream signaling cascades, both ultimately blunt cytokine-mediated endocytosis of cell surface VE-cadherin (15
). Thus, the strength of the endothelial barrier may be a product of a constant tug-of-war between opposing stabilizing and destabilizing signals that control VE-cadherin trafficking, allowing the vascular system the necessary plasticity to respond to changing physiologic needs.
The innate immune system provides the first line of defense of the body against pathogens and must be rapid, broad-spectrum, and toxic to the offending pathogen. There is a fine line between overwhelming the invaders and inflicting severe collateral damage to the host. Boosting the innate immune system with cytokines increases the risk of vasogenic shock, yet efforts to reduce secondary damage by suppressing the innate immune response with glucocorticoids can worsen the outcomes from severe infection. Thus, pharmacologic modulation of the innate immune response has a narrow therapeutic window.
The specific cytokines elaborated and their temporal secretion profile may differ for each pathogen. Nevertheless, the cumulative effects of hypercytokinemia on vascular leakage and noncardiogenic shock are common among severe infections characterized by septic shock (29
). Our data suggest that an alternative approach to combating these infections is to strengthen the vascular barrier. The pharmacologic promotion of vascular stability was sufficient to mute the vascular hyperpermeability induced by multiple different cytokines and the subsequent mortality in rodent models of severe bacterial and viral infections. Enhancing stability offers practical advantages over a more complicated approach aimed at blocking each individual cytokine contributing to cytokine storm. The practical success of targeting the vascular response to cytokines may require a careful characterization of the temporal sequence involving infection, hypercytokinemia, vascular leakage, organ failure, and eventual death (9
). A likely limitation is that this therapeutic approach may need to be used before the vascular damage is too grave to repair. Even so, targeting the host and not the pathogen offers a stratagem that could offer sufficient flexibility to successfully combat ever-changing biologic threats from drug-resistant, mutating, and emerging infectious agents (55