Ischemic Preconditioning Modulates the Peripheral Innate Immune System to Promote Anti-Inflammatory and Protective Responses in Mice Subjected to Focal Cerebral Ischemia

Abstract

The development of tolerance triggered by a sublethal ischemic episode (preconditioning, PC) involves a complex crosstalk between neurons, astrocytes and microglia, although the role of the peripheral immune system in this context is largely unexplored. Here, we report that severe cerebral ischemia caused by transient middle cerebral artery occlusion (MCAo) in adult male mice elevates blood counts of inflammatory neutrophils and monocytes, and plasma levels of miRNA-329-5p. These inflammatory responses are prevented by ischemic PC induced by 15 min MCAo, 72h before the severe insult (1h MCAo). As compared with sham-operated animals, mice subjected to either ischemic PC, MCAo or a combination of both (PC+MCAo) display spleen contraction. However, protein levels of Ym1 (a marker of polarization of myeloid cells towards M2/N2 protective phenotypes) are elevated only in spleen from the experimental groups PC and PC+MCAo, but not MCAo. Conversely, Ym1 protein levels only increase in circulating leukocytes from mice subjected to 1h MCAo, but not in preconditioned animals, which is coincident with a dramatic elevation of Ym1 expression in the ipsilateral cortex. By immunofluorescence analysis, we observe that expression of Ym1 occurs in amoeboid-shaped myeloid cells, mainly representing inflammatory monocytes/macrophages and neutrophils. As a result of its immune-regulatory functions, ischemic PC prevents elevation of mRNA levels of the pro-inflammatory cytokine interleukin (IL)-1β in the ipsilateral cortex, while not affecting IL-10 mRNA increase induced by MCAo. Overall, the elevated anti-inflammatory/pro-inflammatory ratio observed in the brain of mice pre-exposed to PC is associated with reduced brain infarct volume and ischemic edema, and with amelioration of functional outcome. These findings reaffirm the crucial and dualistic role of the innate immune system in ischemic stroke pathobiology, extending these concepts to the context of ischemic tolerance and underscoring their relevance for the identification of novel therapeutic targets for effective stroke treatment.

Characterization of the Involvement of Tumour Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6) in Ischemic Brain Injury Caused by Middle Cerebral Artery Occlusion in Mouse

“…The blots were then incubated overnight at 4 °C, with the following primary antibodies: mouse anti-TSG-6 (1:1000; MABT108, Merck Millipore, Milan, Italy), rabbit anti-Ym1 (1:1000; 60130, StemCell Technologies, Meda, MB, Italy) and mouse anti-β-actin (1:3000; A3853, Sigma-Aldrich, Milan, Italy). This was then followed by incubation with the appropriate secondary antibodies (1:3000; Sigma-Aldrich, Milan, Italy) for 1 h at room temperature [ 13 ]. Immunodetection and quantification of protein bands were performed using the iBright™ FL1500 (Thermo Fisher Scientific, Monza, MB, Italy) and ImageJ software.…”

“…MicroRNA was extracted from plasma samples using a miRNeasy Serum/Plasma Kit (Qiagen, Inc., Hilden, Germany). Briefly, 5 volumes of QIAzol lysis reagent were added to plasma and 5 pM A. Thaliana miR-159a (478411_mir, Life Technologies, Monza, MB, Italy) was spiked into the mixture [ 13 ]. Subsequently, chloroform was mixed in the solution which was then centrifuged for 15 min at 12,000× g at 4 °C to obtain three layers.…”

“…The immune system exerts a dualistic role in the progression of ischemic brain damage, playing detrimental or protective/regenerative roles depending on the specific soluble mediators (e.g., pro- or anti-inflammatory cytokines) or cellular phenotype (e.g., inflammatory M1 or reparative M2) involved [ 9 , 10 , 11 ]. Indeed, recent studies have validated the suitability of a rational immunomodulation by targeting central and/or peripheral immune responses involved in ischemic pathobiology [ 9 , 10 , 12 , 13 ]. Thus, the identification of novel targets to modulate the immune responses triggered by cerebral ischemia represents a desirable aim with strong translational value.…”

Peripheral macrophages in the development and progression of structural cerebrovascular pathologies

“…216,217 While preconditioning responses occur in virtually all cells of the central nervous system, immune cells seem to play a particularly critical role because depleting microglia abolishes preconditioning responses. 218,219 Additionally, reduced inflammation and initiation of vascular remodeling have been described as components of the phenotypic response to preconditioning. 218 A recent study assessing the role of ischemic preconditioning on peripheral macrophages identified an elevated M2/M1 macrophage ratio in the brain following experimental ischemic injury, as well as augmented expression of M2-associated genes.…”

PHLDA1 modulates microglial response and NLRP3 inflammasome signaling following experimental subarachnoid hemorrhage

“…Interestingly, microglia can switch their phenotype under different microenvironments. In a variety of neurological disorders, suppression of microglia M1 polarization and promotion of M2 microglia could effectively reduce acute brain injuries and improve neurological outcomes (9)(10)(11). Thus, modulating microglia M1/M2 polarization might be a feasible method to mitigate EBI.…”