The Role of Macrophage Phenotype and Age in Spinal Cord Injury

Spinal cord injury (SCI) elicits a CNS macrophage response consisting of functionally distinct subsets of classically activated, “M1” (pro-inflammatory) and alternatively activated, “M2” (anti-inflammatory) cells. M1 macrophages are neurotoxic while M2 macrophages promote axon growth and remyelination without concomitant neurotoxicity. Unfortunately, macrophages are polarized toward a M1 phenotype after human and rodent SCI. There is growing published evidence demonstrating that driving M2 macrophage activation significantly improves SCI recovery. The mechanisms underlying the reparative effects of M2 macrophages, however, are not well understood. As a result, the development of clinically viable, pharmacological interventions that harness the reparative potential of activated macrophages remains a critical challenge. The development of immunomodulatory therapies is further challenged by a changing SCI demographic. The incidence of SCI among older individuals has increased in recent years with 61% of all non-fatal SCIs being sustained by individuals >45 years old. With this change in age comes a more equal balance of SCI among both males and females. We have new data that age dampens the M2 response to SCI and published data document that sex steroids can influence the M1/M2 shift of activated microglia and macrophages. Most clinical therapies, however, are being examined in individuals regardless of age or sex and are based upon preclinical data generated almost exclusively using young, female animals. These practices raise concerns about the translational potential of pre-clinical data specifically for immunomodulatory therapies. Our long-term goal is to understand and exploit the naturally reparative functions of CNS macrophages to develop effective immunomodulatory therapies for treating neuropathological conditions. The objective of this proposal is to investigate the pro-reparative mechanisms of M2 macrophages and determine the effect of age and sex on macrophage activation after SCI. Our central hypothesis is that the reparative effects of M2 macrophages depend upon production of arginase-1. Further, we hypothesize that both age and sex affect induction of M2 macrophages after SCI. Arginase production is a hallmark of M2 macrophages. Therefore, our hypothesis is based upon the following published and preliminary observations: 1) both M2 macrophages and arginase facilitate neuroprotection, increase axon regeneration and growth, and promote cell proliferation and remyelination; 2) the macrolide antibiotic azithromycin (AZM) increases M2 macrophagemediated arginase production and AZM treatment improves tissue sparing and functional recovery after SCI; 3) age impairs M2 macrophage activation and macrophage-mediated arginase production in SCI coincident with functional impairments and tissue loss; and 4) female sex hormones potentiate M2 macrophage activation. AIM 1: DETERMINE THE ROLE OF ARGINASE IN M2 MACROPHAGE-MEDIATED REPAIR PROCESSES IN SCI. Based upon observations that both arginase and M2 macrophages regulate similar repair process, we hypothesize that the reparative effects of M2 macrophages are dependent upon arginase production. We will utilize genetically engineered mice with macrophages that lack arginase for loss of function studies and will treat mice with AZM for gain of function studies. Parallel in vivo and in vitro SCI models will be used to evaluate the effects of macrophage-mediated arginase production on neurotoxicity, axon growth/dieback, and remyelination. AIM 2: DETERMINE THE EFFECT OF AGE AND SEX ON THE ACUTE MACROPHAGE RESPONSE TO SCI. Based upon published data that estradiol and progesterone favor M2 activation, we hypothesize that males will have impaired M2 macrophage activation after SCI compared to females. Further, we hypothesize that age will potentiate sex-specific macrophage differences. We will evaluate the macrophage response over time in a clinically relevant mouse model of contusion SCI using a combination of flow cytometry and focused gene profiling. We will utilize 4- and 14-month old male and female mice. AIM 3: DETERMINE THE EFFECT OF AGE AND SEX ON M2 MACROPHAGE-MEDIATED REPAIR AND RECOVERY AFTER SCI. We have observed that AZM drives M2 macrophages in 4 month-old (MO) female SCI mice and AZM treatment improves recovery after SCI. We postulate that the effectiveness of AZM SCI treatment will be reduced as a function of both age and sex. We will test the dose response and therapeutic window of AZM treatment on macrophage phenotype and SCI recovery in 4 and 14 MO male and female mice. Neuroinflammation is a hallmark of most neuropathologies. Therefore, the rationale for the propose research is that, once it is know how M2 macrophages facilitate reparative outcome measures and are regulated by different physiological conditions, new and innovative pharmacological approaches can be developed with positive translational impact for treating not only SCI, but a variety of neuroinflammatory conditions.