An injection of nanoparticles can prevent the body’s immune system from overreacting to trauma, potentially preventing some spinal cord injuries from resulting in paralysis.
The approach was demonstrated in mice at the University of Michigan, with the nanoparticles enhancing healing by reprogramming the aggressive immune cells – call it an “EpiPen” for trauma to the central nervous system, which includes the brain and spinal cord.
“In this work, we demonstrate that instead of overcoming an immune response, we can co-opt the immune response to work for us to promote the therapeutic response,” said Lonnie Shea, the Steven A. Goldstein Collegiate Professor of Biomedical Engineering.
Trauma of any kind kicks the body’s immune response into gear.
In a normal injury, immune cells infiltrate the damaged area and clear debris to initiate the regenerative process.
The central nervous system, however, is usually walled off from the rough-and-tumble of immune activity by the blood-brain barrier.
A spinal cord injury breaks that barrier, letting in overzealous immune cells that create too much inflammation for the delicate neural tissues.
That leads to the rapid death of neurons, damage to the insulating sheaths around nerve fibers that allow them to send signals, and the formation of a scar that blocks the regeneration of the spinal cord’s nerve cells.
All of this contributes to the loss of function below the level of the injury.
That spectrum includes everything from paralysis to a loss of sensation for many of the 12,000 new spinal injury patients each year in the United States.
Previous attempts to offset complications from this immune response included injecting steroids like methylprednisolone.
That practice has largely been discarded since it comes with side effects that include sepsis, gastrointestinal bleeding and blood clots.
The risks outweigh the benefits.
But now, U-M researchers have designed nanoparticles that intercept immune cells on their way to the spinal cord, redirecting them away from the injury.
Those that reach the spinal cord have been altered to be more pro-regenerative.
With no drugs attached, the nanoparticles reprogram the immune cells with their physical characteristics: a size similar to cell debris and a negative charge that facilitates binding to immune cells.
In theory, their nonpharmaceutical nature avoids unwanted side effects.
With fewer immune cells at the trauma location, there is less inflammation and tissue deterioration.
Second, immune cells that do make it to the injury are less inflammatory and more suited to supporting tissues that are trying to grow back together.
“Hopefully, this technology could lead to new therapeutic strategies not only for patients with spinal cord injury but for those with various inflammatory diseases,” said Jonghyuck Park, a U-M research fellow working with Shea.
Previous research has shown success for nanoparticles mitigating trauma caused by the West Nile virus and multiple sclerosis, for example.
“The immune system underlies autoimmune disease, cancer, trauma, regeneration—nearly every major disease,” Shea said.
“Tools that can target immune cells and reprogram them to a desired response have numerous opportunities for treating or managing disease.”
Intravenously infused synthetic 500 nm nanoparticles composed of poly(lactide-co-glycolide) are taken up by blood-borne inflammatory monocytes via a macrophage scavenger receptor (macrophage receptor with collagenous structure), and the monocytes no longer traffic to sites of inflammation.
Intravenous administration of the nanoparticles after experimental spinal cord injury in mice safely and selectively limited infiltration of hematogenous monocytes into the injury site.
The nanoparticles did not bind to resident microglia, and did not change the number of microglia in the injured spinal cord.
Nanoparticle administration reduced M1 macrophage polarization and microglia activation, reduced levels of inflammatory cytokines, and markedly reduced fibrotic scar formation without altering glial scarring.
These findings thus implicate early-infiltrating hematogenous monocytes as highly selective contributors to fibrosis that do not play an indispensable role in gliosis after SCI.
Further, the nanoparticle treatment reduced accumulation of chondroitin sulfate proteoglycans, increased axon density inside and caudal to the lesion site, and significantly improved functional recovery after both moderate and severe injuries to the spinal cord.
These data provide further evidence that hematogenous monocytes contribute to inflammatory damage and fibrotic scar formation after spinal cord injury in mice.
Further, since the nanoparticles are simple to administer intravenously, immunologically inert, stable at room temperature, composed of an FDA-approved material, and have no known toxicity, these findings suggest that the nanoparticles potentially offer a practical treatment for human spinal cord injury.
Introduction
Traumatic injury to the spinal cord (SCI) disrupts the blood-brain barrier and leads to a cascade of secondary responses including a rapid influx of monocytes into the injured area. Monocyte infiltration occurs in a biphasic pattern (Berton and Lowell, 1999; Boros et al., 2010; Shantsila et al., 2011) that begins within hours after SCI.
The number of macrophages peaks at 7 days with a second peak at 14–28 days post injury. Infiltrating monocytes, as well as tissue resident microglia, differentiate into macrophages (Fleming et al., 2006; Beck et al., 2010; David and Kroner, 2011). Monocytes and macrophages/microglia in the injured spinal cord have both detrimental and beneficial actions, and the exact roles of these populations after SCI are yet to be fully elucidated (Meda et al., 1995; Popovich et al., 1999; Popovich et al., 2002; Majed et al., 2006; Letellier et al., 2010). Recent studies suggest that the early influx of hematogenously-derived macrophages (hMΦ), but not macrophages derived from resident microglia (mMΦ), is primarily responsible for secondary axonal dieback after SCI (Beck et al., 2010; Durafourt et al., 2012; Evans et al., 2014; Gensel and Zhang, 2015). Thus, selectively blocking hMΦ infiltration during the early phase of SCI without altering microglia could help limit secondary tissue damage while preserving the beneficial effects of mMΦ.
The tools used in prior studies of monocyte/macrophage depletion do not exclusively target only hMΦ. Clodronate liposomes, when injected intravenously, deplete circulating monocytes and improve motor functions after SCI in rodents (Popovich et al., 1999; Horn et al., 2008; Grosso et al., 2014). However, clodronate liposomes also target and destroy CNS resident microglia (Kumamaru et al., 2012; Plemel et al., 2014), which may reduce the beneficial effects exerted by microglia in the damaged spinal cord. CCR2 antagonists (Kang et al., 2011), and CCR2 small interfering RNA (Leuschner et al., 2011) target hematogenous monocytes and may spare microglia which do not express CCR2 (Jung et al., 2009; Mizutani et al., 2012). However, this approach also targets T cells and immature B cells that express CCR2 (Mack et al., 2001; Flaishon et al., 2004). There are similar issues with other techniques that have been used to reduce macrophage infiltration after SCI (Mabon et al., 2000; Fiore et al., 2004; Stirling et al., 2004; Lopez-Vales et al., 2005).
Here, we sought to selectively deplete hΦM after SCI by using intravenously injected biodegradable carboxylated poly(lactide-co-glycolide) (PLGA) immune-modifying nanoparticles (IMPs).
IMPs are highly negatively charged, synthetic, 500 nm-diameter particles that bind to the macrophage receptor with collagenous structure (MARCO) on monocytes. IMPs are immunologically inert and simple to manufacture (Getts et al., 2012).
We chose to use 500 nm diameter particles because a previous study reported that 500 nm diameter microparticles have a higher binding affinity for MARCO than microparticles with 20 nm, 200 nm, or 1000 nm diameters (Sanae Kanno and Hirano, 2007).
Monocytes bound to IMPs no longer travel to sites of inflammation, but instead are sequestered in the spleen where they undergo cas-pase-3 mediated apoptosis (Getts et al., 2014).
IMPs reduce tissue damage and improve outcomes in animal models of several inflammatory diseases including encephalomyelitis, lethal flavivirus encephalitis, myocardial infarction, dextran sodium sulfate–induced colitis, and thioglycollate-induced peritonitis (Getts et al., 2012; Getts et al., 2014).
SCI leads to scarring at the lesion site that includes both fibrotic and gliotic responses (Goritz et al., 2011; Soderblom et al., 2013; Zhu et al., 2015a; Zhu et al., 2015b).
The lesional scar inhibits axonal regeneration through a number of mechanisms including accumulation of molecules that are inhibitory to axonal outgrowth, such as chondroitin sulfate proteoglycans (CSPGs), and acting as a physical impediment to axon elongation.
The relative roles of gliosis and fibrosis in inhibiting axon outgrowth are unclear.
Traditionally astrogliosis has been viewed as an impediment to axon outgrowth, but some evidence suggests that certain populations of astrocytes could enhance regeneration after SCI (Bush et al., 1999; Faulkner et al., 2004; Anderson et al., 2016).
The fibrotic response appears to arise from perivascular fibroblasts, which secrete the majority of fibronectin in spinal lesions (Goritz et al., 2011; Soderblom et al., 2013; Zhu et al., 2015a; Zhu et al., 2015b). Secreted fibronectin dimers are then assembled into an insoluble fibronectin matrix via an integrin dependent mechanism.
The assembled matrix is characterized by abundantly crosslinked fibronectin that fails to be successfully remodeled and has been shown to remain even at chronic time points after SCI (Zhu et al., 2015b). In toto these observations suggest that both gliosis and fibrosis at the lesion site influence regenerative responses after SCI.
In this paper, we report that IMPs administered intravenously (iv) after SCI significantly reduced numbers of intralesional inflammatory macrophages and other hematogenous inflammatory cells and decreased the proportion of M1-polarized inflammatory macrophages.
IMPs did not bind to resident microglia, and IMPs treatment after SCI did not change the number of microglia in the injured spinal cord. hMΦ depletion via IMPs treatment diminished fibrotic scarring and collagen accumulation in the injured sites, suggesting a relationship between hematogenous monocytes and chronic fibrotic scarring.
IMPs treatment did not alter glial scarring but did reduce accumulation of chondroitin sulfate proteoglycans in the scar. Moreover, IMPs treatment significantly improved recovery of motor function in mouse models of both moderate and severe SCI.
IMPs are simple to administer intravenously, are stable at room temperature for up to several months, are composed of an FDA-approved material and have no known animal toxicity. The outstanding translatability of IMPs, combined with our animal data, suggests that IMPs potentially offer a practical treatment for human SCI.
More information: Jonghyuck Park et al, Intravascular innate immune cells reprogrammed via intravenous nanoparticles to promote functional recovery after spinal cord injury, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1820276116
Journal information: Proceedings of the National Academy of Sciences
Provided by University of Michigan
