Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure

Sanchez et al. Acta Neuropathologica Communications https://doi.org/10.1186/s40478-019-0701-y (2019) 7:54 RESEARCH Open Access Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure Joshua J. Sanchez1, Jacob E. Sanchez1, Shahani Noor1, Chaselyn D. Ruffaner-Hanson1, Suzy Davies1, Carston R. Wagner4, Lauren L. Jantzie1,3, Nikolaos Mellios1, Daniel D. Savage1,3 and Erin D. Milligan1,2* Abstract Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral neuropathy following minor nerve injury. This effect coincides with elevated spinal cord astrocyte activation and ex vivo immune cell reactivity assessed by proinflammatory cytokine interleukin (IL) -1β protein expression. Additionally, the β2-integrin adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1), a factor that influences the expression of the proinflammatory/anti-inflammatory cytokine network is upregulated. Here, we examine whether PAE increases the proinflammatory immune environment at specific anatomical sites critical in the pain pathway of chronic sciatic neuropathy; the damaged sciatic nerve (SCN), the dorsal root ganglia (DRG), and the spinal cord. Additionally, we examine whether inhibiting LFA-1 or IL-1β actions in the spinal cord (intrathecal; i.t., route) could alleviate chronic neuropathic pain and reduce spinal and DRG glial activation markers, proinflammatory cytokines, and elevate anti-inflammatory cytokines. Results show that blocking the actions of spinal LFA-1 using BIRT-377 abolishes allodynia in PAE rats with sciatic neuropathy (CCI) of a 10 or 28-day duration. This effect is observed (utilizing immunohistochemistry; IHC, with microscopy analysis and protein quantification) in parallel with reduced spinal glial activation, IL-1β and TNFα expression. DRG from PAE rats with neuropathy reveal significant increases in satellite glial activation and IL-1β, while IL-10 immunoreactivity is reduced by half in PAE rats under basal and neuropathic conditions. Further, blocking spinal IL-1β with i.t. IL-1RA transiently abolishes allodynia in PAE rats, suggesting that IL-1β is in part, necessary for the susceptibility of adult-onset peripheral neuropathy caused by PAE. Chemokine mRNA analyses from SCN, DRG and spinal cord reveal that increased CCL2 occurs following CCI injury regardless of PAE and BIRT-377 treatment. These data demonstrate that PAE creates dysregulated proinflammatory IL-1β and TNFα /IL-10 responses to minor injury in the sciatic-DRG-spinal pain pathway. PAE creates a risk for developing peripheral neuropathies, and LFA-1 may be a novel therapeutic target for controlling dysregulated neuroimmune actions as a consequence of PAE. Keywords: Neuropathic pain, Prenatal alcohol exposure, Glia, Neuroimmune function, Peripheral immune system, Spinal cord * Correspondence: 1 Department of Neurosciences, School of Medicine, University of New Mexico Health Sciences Center, MSC08 4740, Albuquerque, NM 87131-0001, USA 2 Department of Anesthesiology and Critical Care Medicine, University of New Mexico Health Sciences Center, MSC08 4740, Albuquerque, NM 87131-0001, USA Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sanchez et al. Acta Neuropathologica Communications (2019) 7:54 Page 2 of 22 Introduction Fetal alcohol spectrum disorder (FASD), a condition that occurs as a result of prenatal alcohol exposure (PAE), results in cognitive and behavioral deficits [32]. Unfortunately, many of the central nervous system (CNS) deficits are not always apparent. By using animal models of FASD, many of the insidious effects of PAE have been uncovered. Interestingly, a growing body of evidence shows that PAE alters immune function and reactivity. Specifically, the viability of microglial cells within the cerebellum is decreased as a consequence of developmental ethanol exposure [22]. Additionally, a separate study demonstrates that hippocampal glial cells are increased in their activation state, which coincides with heightened expression of the proinflammatory cytokine interleukin (IL)-1β [57]. These and other PAE studies utilize high levels of alcohol exposure to mimic the effects of binge alcohol consumption, leaving the question of whether low-to-moderate levels of PAE adversely impact the function of the central nervous system in adulthood. Recent reports investigated whether spinal cord pain processing is exaggerated by the effects of PAE on glial and proinflammatory cytokine actions. These studies demonstrate that moderate PAE enhances both peripheral and spinal immune cell and proinflammatory cytokine action. That is, immune responses become ‘primed’ (i.e. sensitized) such that applying a sciatic nerve (SCN) injury generates potentiated microglial and astrocyte responses at the spinal cord, as well as enhanced expression of proinflammamtory cytokines, such as tumor necrosis factor-α (TNFα) and IL-1β, at the damaged SCN [41, 42]. Additionally, blunted expression of the anti-inflammatory cytokine IL-10, which suppresses neuropathic pain [24, 61], is observed in the dorsal root ganglia (DRG) and the SCN in PAE rats compared to non-PAE controls [42]. These results support the possibility that PAE results in susceptibility to pro-nociceptive proinflammatory neuroimmune responses well into adulthood. In an effort to address the possible risk of developing neuropathy created by PAE, the injury to the SCN was substantially reduced in an effort to unmask the effects of moderate PAE on glial and immune priming [49]. This reduction uncovered an alteration in spinal astrocytes, as the astrocyte marker, glial fibrillary acidic protein (GFAP) is significantly elevated in PAE rats with neuropathy [42, 49], while evidence for microglial activation was not observed. In support of a role for peripheral immune cells in sensitized responses to minor injury/challenge, heightened expression of TNFα and IL-1β was observed in leukocytes from PAE rats [49]. Together, these data suggest that the PAE leads to hyper-reactive neuroimmune response which may render one vulnerable to developing neuropathic pain. Interestingly, the chemokine CCL2 and the β2-integrin, lymphocyte function-associated antigen-1 (LFA-1) are elevated in PAE rats [42]. CCL2 activates its cognate recepto (...truncated)