Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes

Abstract

BackgroundNeutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.MethodsEAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.ResultsWe discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.ConclusionsThese results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

“…Importantly, we also validated that the collagenase D + DNase I digestion does not affect the detection of numerous immune markers (KLRG1, NKG2D, TCR beta chain, CD44, CD62L, NK1.1, CD127, TCR gamma/delta) by comparing splenocytes from undigested vs. collagenase D + DNase I digested organs (data not shown). As expected and observed in numerous studies of CNS injury models (EAE/neuropathic pain) (Berghmans et al, 2012;Cao et al, 2012), the number of hematopoietic cells was dramatically increased in EAE mice (3.8 millions) compared to naïve animals (310,000 cells) (Fig. 4).…”

“…Some groups did not enzymatically digest CNS tissues (Berghmans et al, 2012;Cao et al, 2012); others digested samples with trypsin (Phares et al, 2009). Moreover, these groups got rid of myelin and cell debris using the Percoll TM gradient step (Berghmans et al, 2012;Cao et al, 2012;Jin et al, 2007;Phares et al, 2009).…”

“…In several studies, experimental animals have been pooled to obtain sufficient numbers of cells to perform flow cytometry analysis (Berghmans et al, 2012;Cao et al, 2012;Phares et al, 2009). Some groups did not enzymatically digest CNS tissues (Berghmans et al, 2012;Cao et al, 2012); others digested samples with trypsin (Phares et al, 2009).…”

“…Embryonic rodent tissues from the central nervous system (CNS) can be efficiently digested and analyzed by flow cytometry (Jungblut et al, 2012;Panchision et al, 2007). For more than two decades, different groups have elaborated strategies to isolate microglia and infiltrating leukocytes from adult rodent CNS tissue despite their high lipidic content (Berghmans et al, 2012;Campanella et al, 2002;Cardona et al, 2006;Dick et al, 1995;Ford et al, 1995;Havenith et al, 1998;Pino and Cardona, 2011;Saura et al, 2003;Sedgwick et al, 1991). Combinations of mechanical and enzymatic digestions (e.g.…”

Central nervous system diseases and the role of the blood-brain barrier in their treatment

“…This allows leukocytes to enter into the brain serving to speed a neuroinflammatory cascade. Although the causes of both AD and PD remain unknown, patterns of familial inheritance suggest a possible connection involving abnormal protein processing (Aβ for AD and alpha synuclein for PD) and accumulation [22].…”