How Inflammation May Impact LNB Patients

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
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RitaA
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Joined: Thu 1 Jul 2010 8:33

How Inflammation May Impact LNB Patients

Post by RitaA » Mon 30 Apr 2012 9:32

http://www.jneuroinflammation.com/conte ... 4-9-72.pdf
Journal of Neuroinflammation

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A possible role for inflammation in mediating apoptosis of oligodendrocytes as induced by the Lyme disease spirochete Borrelia burgdorferi

Journal of Neuroinflammation 2012, 9:72 doi:10.1186/1742-2094-9-72

Geeta Ramesh (gramesh@tulane.edu)
Shemi Benge (abenge@tulane.edu)
Bapi Pahar (bpahar@tulane.edu)
Mario T Philipp (philipp@tulane.edu)

ISSN 1742-2094

Article type Research
Submission date 10 February 2012
Acceptance date 23 April 2012
Publication date 23 April 2012

Article URL http://www.jneuroinflammation.com/content/9/1/72

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© 2012 Ramesh et al. ; licensee BioMed Central Ltd.

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A possible role for inflammation in mediating
apoptosis of oligodendrocytes as induced by the
Lyme disease spirochete Borrelia burgdorferi

Geeta Ramesh1
Email: gramesh@tulane.edu
Shemi Benge2
Email: abenge@tulane.edu
Bapi Pahar3
Email: bpahar@tulane.edu
Mario T Philipp1*
* Corresponding author
Email: philipp@tulane.edu

1 Division of Bacteriology and Parasitology, Tulane National Primate Research
Center, Covington, LA, USA
2 School of Science and Engineering, Tulane University, New Orleans, LA, USA
3 Division of Comparative Pathology, Tulane National Primate Research Center,
Covington, LA, USA

Abstract

Background

Inflammation caused by the Lyme disease spirochete B. burgdorferi is an important factor in
the pathogenesis of Lyme neuroborreliosis. Our central hypothesis is that B. burgdorferi can
cause disease via the induction of inflammatory mediators such as cytokines and chemokines
in glial and neuronal cells. Earlier we demonstrated that interaction of B. burgdorferi with
brain parenchyma induces inflammatory mediators in glial cells as well as glial
(oligodendrocyte) and neuronal apoptosis using ex vivo and in vivo models of
experimentation.

Methods

In this study we evaluated the ability of live B. burgdorferi to elicit inflammation in vitro in
differentiated human MO3.13 oligodendrocytes and in differentiated primary human
oligodendrocytes, by measuring the concentration of immune mediators in culture
supernatants using Multiplex ELISA assays. Concomitant apoptosis was quantified in these
cultures by the in situ terminal deoxynucleotidyl transferase mediated UTP nick end labeling
(TUNEL) assay and by quantifying active caspase-3 by flow cytometry. The above
phenomena were also evaluated after 48 h of stimulation with B. burgdorferi in the presence
and absence of various concentrations of the anti-inflammatory drug dexamethasone.

Results

B. burgdorferi induced enhanced levels of the cytokine IL-6 and the chemokines IL-8 and
CCL2 in MO3.13 cells as compared to basal levels, and IL-8 and CCL2 in primary human
oligodendrocytes, in a dose-dependent manner. These cultures also showed significantly
elevated levels of apoptosis when compared with medium controls. Dexamethasone reduced
both the levels of immune mediators and apoptosis, also in a manner that was dose
dependent.

Conclusions

This finding supports our hypothesis that the inflammatory response elicited by the Lyme
disease spirochete in glial cells contributes to neural cell damage. As oligodendrocytes are
vital for the functioning and survival of neurons, the inflammation and subsequent apoptosis
of oligodendrocytes induced by B. burgdorferi could contribute to the pathogenesis of Lyme
neuroborreliosis.




Background

Lyme neuroborreliosis (LNB) in the US is manifest in 10% to 15% of patients diagnosed
with Lyme disease [1,2]. In addition to the classical neurological triad of meningitis, cranial
neuritis, and radiculitis, LNB may also manifest, albeit more rarely, as encephalopathy,
encephalomyelitis [3,4], and cerebellitis [5]. Acute transverse myelitis, caused by
inflammatory processes of the spinal cord resulting in axonal demyelination, has also been
reported in LNB patients [6-9]. In the peripheral nervous system (PNS), Lyme disease
appears as neuritis with patchy multifocal axonal degeneration associated with epineural
perivascular inflammation [10,11].

LNB patients may experience a wide array of neurological and neuropsychiatric symptoms as
a result of white matter inflammation that results in a subacute multiple sclerosis (MS)-like
manifestation [12,13]. Brain magnetic resonance imaging (MRI) of LNB patients that was
suggestive of a demyelinating disease, with MS-like symptoms that responded well to
antibiotic therapy, has been reported [14,15]. It has been hypothesized that B. burgdorferi
may exacerbate MS or be a trigger for an MS-like inflammatory demyelinating disease of the
central nervous system (CNS) by activating myelin-specific T cells via molecular mimicry
[16,17], or by bystander activation via inflammatory cytokines [16].

Encephalitis associated with LNB involves white matter more often than gray matter
[4,18,19]. Inflammatory lesions in the brain and spinal cord show multifocal encephalitis
with large areas of demyelination in perivascular white matter commonly associated with the
presence of B. burgdorferi DNA [6,20-22]. Astroglial and neuronal proteins, anti-myelin
antibodies and cells secreting antibodies to myelin basic protein have been detected in the
cerebrospinal fluid (CSF) of patients with LNB, indicating possible glial and neuronal
damage in the CNS parenchyma [23-25]. There is evidence that B. burgdorferi spirochetes
can adhere to neurons, CNS glia, and Schwann cells from studies in neuronal and glial cell
lines and primary rat brain cultures [26], and that B. burgdorferi can adhere to and perhaps
invade human neuroglial and cortical neuronal cells [27]. Adhesion was found to be
associated with galactocerebroside, a glycolipid component of myelin, and oligodendrocytes
in primary brain cultures were shown to be damaged, by scanning electron microscopy
[26,28,29]. Cells that secrete antibodies to myelin basic protein have been found in CSF of
patients with LNB, suggesting damage to oligodendrocytes possibly as a result of
demyelination [24].

Cytokines and chemokines are key immune mediators that play an important role in
promoting CNS injury in various kinds of inflammatory neurodegenerative diseases [30-34].
Various inflammatory cytokines and chemokines have been reported in the CSF of patients
with LNB [35-38].

We hypothesize that B. burgdorferi can cause disease via the induction of inflammatory
mediators such as cytokines and chemokines in glial and neuronal cells. Earlier we
demonstrated that interaction of B. burgdorferi with brain parenchyma induces inflammatory
mediators in glial cells as well as glial (oligodendrocyte) and neuronal apoptosis [39].
Further, we found that a similar inflammatory response occurs in vivo, as demonstrated in
rhesus monkeys inoculated intrathecally with live B. burgdorferi. This resulted in elevation of
IL-6, IL-8, CCL2, and CXCL13 in the CSF within 1 week post infection, accompanied with
histopathological changes consistent with acute neurological Lyme disease such as
leptomeningitis and radiculitis, as well as satellite glial cell and neuronal apoptosis in the
dorsal root ganglia [40].

Here we assessed the ability of live B. burgdorferi to elicit inflammatory mediators in
cultures of differentiated human MO3.13 oligodendrocytes [41], and primary cultures of
differentiated human oligodendrocyte precursor cells (HOPC). Further, we examined the
ability of live B. burgdorferi to induce apoptosis of oligodendrocytes, and quantified
apoptosis in the above cultures by the in situ TUNEL assay, and by measuring activated
caspase-3 by flow cytometry. The role of inflammation in mediating apoptosis of
oligodendrocytes, as induced by B. burgdorferi was studied by evaluating the above
phenomena after 48 h of stimulation with B. burgdorferi in the presence and absence of
various concentrations of the anti-inflammatory drug dexamethasone, a glucocorticoid used
in the treatment of immune-mediated inflammatory diseases [42].

[big snip]

Discussion

In recent studies, using ex vivo and in vivo modes of experimentation in the rhesus monkey
model of LNB, we had established that B. burgdorferi is able to induce inflammatory
mediators, with concomitant apoptosis of oligodendrocytes in the frontal cortex, and of
satellite glial cells in dorsal root ganglia [39,40]. We had also shown with experiments
performed in vitro that human neurons co-cultured with B. burgdorferi and rhesus microglia
undergo apoptosis in the presence of pro-inflammatory mediators chiefly produced by the
microglia [44]. In this study we focused on evaluating the ability of live B. burgdorferi to
induce oligodendrocyte damage in an in vitro system, using differentiated MO3.13 human
oligodendrocytes and differentiated HOPC. We addressed the hypothesis that inflammation
plays a role in mediating apoptosis of oligodendrocytes, as induced by B. burgdorferi, using
the anti-inflammatory drug dexamethasone. We included HOPC in our study to corroborate
the observations that we made with the MO3.13 cell line.

We established in vitro cultures of MO3.13 cells and confirmed the presence of phenotypic
markers that are known to be expressed by this cell line, namely MBP and GFAP [41].
Our first key observation was that B. burgdorferi is able to induce the pro-inflammatory
mediators CCL2, IL-6, and IL-8 in oligodendrocytes. The levels of immune mediators
detected in the culture supernatants increased concordantly with an increase in the spirochetal
MOI. HOPC similarly produced CCL2 and IL-8, a finding that further validates the results
obtained with MO3.13 cells. These observations echo our previous findings made with
astrocytes and microglia, as these glial cells also produced pro-inflammatory mediators in
response to live B. burgdorferi, and expand the scope of our hypothesis of a role for glial
cells in mediating inflammation in LNB [39,40,44-46]. Oligodendrocytes could therefore
contribute to the elevated levels of cytokines and chemokines detected in the CSF of patients
with LNB [35-38].

Cytokines and chemokines play a central role in inflammation, demyelination, and
neurodegeneration in the CNS during inflammatory neurodegenerative diseases such as
multiple sclerosis (MS) [47]. Oligodendrocytes in brain tissue that is immediately adjacent to
the subarachnoid space, the region known as the sub-pial space, are especially vulnerable to
demyelination [48]. Since inflammatory lesions are commonly found in the meninges in
LNB, the myelitis that is seen in LNB may be in part due to oligodendrocytes. These cells
could be damaged by the inflammatory process brought about by the oligodendrocytes
themselves, with participation of other glial cells, in addition to inflammatory mediators
produced by the perivascular cellular infiltrates that are often present in CNS infection.
Oligodendrocytes are known to express receptors for various cytokines and chemokines [49].
CCL2 was induced at high levels in oligodendrocytes by B. burgdorferi. This chemokine is of
particular importance in mediating inflammation in neurodegenerative diseases [50]. CCL2
recruits monocytes and T cells from the blood stream into the CNS during acute
neuroinflammation, in addition to recruiting microglia, the resident macrophages of the brain
[51]. It is an important mediator in many neuroinflammatory and neurodegenerative brain
diseases characterized by neuronal degeneration [52]. CCL2 has been found to be upregulated
in actively demyelinating MS plaques [53], and its expression is increased in
experimental autoimmune encephalomyelitis [54]. It is known to modulate microglial
activation and proliferation, thus contributing to the inflammatory response mounted by the
CNS [55]. Importantly, CCL2 levels are elevated in the CSF of patients with LNB [56], and
we found high levels of CCL2 in the CSF of rhesus monkeys infected intrathecally with B.
burgdorferi [40]. CCL2 also has been documented to play a role in mediating nerve damage
and demyelination of axons by causing influx of monocytes and T cells, in Wallerian
degeneration [57,58], and may thus contribute to the axonal damage that affects patients with
LNB of the PNS [10,11].

The cytokine IL-6, which was also elevated in the culture supernatants of oligodendrocytes
that were exposed to live B. burgdorferi, is known to be both helpful and harmful in the CNS
[31-34]. Dysregulated expression of IL-6 has been documented in several neurological
disorders such as MS, acute transverse myelitis, Alzheimer’s disease, schizophrenia, epileptic
seizures, and Parkinson’s disease [49]. In addition, IL-6 has been shown to be involved in
multiple physiological CNS processes such as neuron homeostasis, astrogliogenesis, and
neuronal differentiation [59]. Elevated levels of IL-6 have also been found in the CSF of
LNB patients [35]. IL-6 is known to promote oligodendrocyte and neuronal survival in the
presence of glutamate-mediated excitotoxicity in hyppocampal slices [60]. IL-6 is also known
to support survival of oligodendrocytes in vitro [61].

The third pro-inflammatory mediator whose concentration was significantly increased in
culture supernatants of oligodendrocytes stimulated with live B. burgdorferi is IL-8. This
chemokine also has been reported to be elevated in the CSF of LNB patients [62]. We had
previously documented that B. burgdorferi induces production of IL-8 in rhesus microglia,
astrocytes and endothelial cells [39,40,44,46]. IL-8 released into the CSF after brain injury is
associated with blood-brain barrier dysfunction and plays a central role in recruitment of
neutrophils and T cells into the CNS during bacterial meningitis [63,64].

Our second key observation was that live B. burgdorferi induce a significantly elevated level
of apoptosis, as assessed by the TUNEL assay, in MO3.13 oligodendrocytes compared to that
seen in medium controls. The level of apoptosis observed increased concordantly with an
increase in the B. burgdorferi MOI. We also observed elevated levels of activated caspase-3,
a phenomenon that is known to be an early signaling event that results in apoptosis [65]. The
MO3.13 oligodendrocyte cell line used in these studies has also been shown to undergo
active caspase-3-mediated apoptosis due to other stimuli such as ceramide [66,67], and
inflammatory cytokines [68]. Caspase-1, -2 and -3 are known to be expressed in mature
oligodendrocytes [69]. Caspase-mediated oligodendrocyte cell death (particularly via
activation of caspase-11 and caspase-3) has also been documented in inflammatory
demyelinating diseases such as MS [70].

The interaction of B. burgdorferi with oligodendrocytes resulted in elevated levels of
inflammatory mediators and concomitant apoptosis in oligodendrocytes, suggesting that the
phenomena of inflammation and apoptosis might be causally related. To uncover the possible
connection between inflammation and apoptosis in this system we treated both differentiated
MO3.13 cells as well as differentiated HOPC with the anti-inflammatory drug
dexamethasone. In both cases the effect was not only a reduction in the amount of proinflammatory
mediators, as would be expected in the presence of dexamethasone, but also a
significant reduction in the fraction of cells undergoing apoptosis. This outcome is a strong
indication that inflammation plays a role in mediating oligodendrocyte apoptosis.
Cytokines such as TNF, IL-1β, lymphotoxin (LT), and TGF-β are known to cause cell death
in oligodendrocytes [71-74]. TNF and IL-1β were not detected in the culture supernatants of
oligodendrocytes that were incubated with live B. burgdorferi for 48 h. TGF-β and LT were
not among the mediators that were detected by the human 14-plex array that we used and
may well have been present in the culture supernatants. TNF, LT, [71] and TGF-β [72] were
shown to induce apoptosis in oligodendrocytes when added exogenously, while IL-1β caused
glutamate-mediated excitotoxic death of oligodendrocytes co-cultured with astrocytes and
microglia [73], or when injected intra-cerebrally in neonatal rats [74].

The potential of CCL2, IL-6, and/or IL-8 to induce oligodendrocyte apoptosis has not been
documented thus far in the literature. In fact, IL-6 is known to promote the survival of
oligodendrocytes in culture [61]. IL-8 has been shown to induce the expression of proinflammatory
proteases, matrix metalloproteinases MMP-2 and MMP-9, cell-cycle protein
cyclin D1, an early marker for G1/S transition and pro-apoptotic protein Bim (Bcl-2-
interacting mediator of cell death), and cell death in cultured neurons in 24 h [75]. CCL2 is
implicated in mediating oligodendrocyte/white matter damage indirectly by mediating the
influx of immune cells such as T cells and macrophages, resulting in cytotoxic damage of the
myelin sheath of axons, followed by phagocytosis of myelin debris, culminating in
demyelination and axonal damage [76]. A possible involvement of cytotoxic cells in the
immune response against B. burgdorferi has been suggested based on in vitro studies [77], in
addition to reports indicating the presence of a cytolytic phenotype of IFN-γ producing cells
from patients with LNB [78]. It is likely that a similar mechanism may be mediating the
demyelination and axonal degeneration resulting in white matter lesions seen in LNB [4,6,18-
22].

The anti-inflammatory effect of dexamethasone, a glucocorticoid used in the treatment of
immune-mediated inflammatory diseases is well documented [42]. Dexamethasone has been
shown to effectively reduce the levels of IL-6, IL-1β, and TNF released from human
monocytes stimulated with endotoxin to below background levels [79]. Dexamethasone
reduced the levels of CCL2 in brain and retinal vascular endothelial cells that were activated
with pro-inflammatory cytokines IL-1β, TNF, and IFN-γ [80]. The anti-inflammatory
potential of dexamethasone to reduce CCL2 and IL-8 also has been reported in cultured
rheumatoid synoviocytes [81]. Here we show that dexamethasone can reduce the levels of
CCL2, IL-6, and IL-8 as induced by B. burgdorferi in differentiated human oligodendrocytes.
Clinical improvement was seen in a severe case of neuroborreliosis showing
encephalomyelitis with polyneuropathy, when treated with the classically recommended 2 to
4 weeks of anti-microbial agents in combination with steroids [82].

Dexamethasone has been shown to suppress CCL2 production via mitogen-activated protein
kinase phosphatase-1 (MAPK-P1)-dependent inhibition of Jun N-terminal kinase and p38
MAPK in activated rat microglia [43]. MAPK cascades are signal transduction pathways that
play important regulatory roles in the biosynthesis of pro-inflammatory cytokines such as IL-
6, IL-8, and CCL2 [83]. MAKP-P1, a member of the Map Kinase Phosphatase family, is
essential for the dephosphorylation/deactivation of MAPK p38 and JNK, thereby limiting
pro-inflammatory cytokine biosynthesis in innate immune cells exposed to microbial
components or infectious agents [84]. MAPK such as p38 and JNK may be involved in the
signaling mechanisms underlying both inflammation and apoptosis [83,85]. Earlier we had
documented the role of p38 MAPK, Erk1, and Erk 2 in mediating the production of IL-6 and
TNF, as well as apoptosis, in rhesus astrocytes as induced by lipoproteins of B. burgdorferi
[86]. MAPK signaling pathways may indeed be involved in regulating both inflammation and
apoptosis as induced by B. burgdorferi in human oligodendrocytes, as well as in the
modulatory effect of dexamethasone that we observed.

Conclusions

In this study we have established that live B. burgdorferi are capable of eliciting
inflammatory mediators, particularly IL-6, IL-8, and CCL2, in addition to inducing apoptosis
in human oligodendrocyte cultures in vitro, by activating caspase-3. Oligodendrocytes are the
myelinating cells of the CNS that myelinate neuronal axons, providing saltatory conduction
of action potentials and proper function of the CNS [87]. The role of oligodendrocyte death in
MS is well established [88]. Some of the earliest pathological changes in inflammatory
lesions seen in MS are increases in oligodendrocyte apoptosis [89,90]. Based on the
observations of this study we propose that neurologic injury in the CNS during an infection
with the Lyme disease spirochete B. burgdorferi could be mediated in part by the direct
action of the spirochetes on oligodendrocytes or via inflammation mediated by B. burgdorferi
in oligodendrocytes. As oligodendrocytes are vital for the survival and optimum function of
neurons [91], oligodendrocyte damage could contribute to neuronal dysfunction and death
and result in the impairment of CNS functions that are seen in patients with LNB.

slmo
Posts: 8
Joined: Tue 10 Apr 2012 17:31

Re: How Inflammation May Impact LNB Patients

Post by slmo » Tue 1 May 2012 10:24

Thank you for this fascinating article. In much research, MS recurs as Lyme in disguise. That in mind, if MS patients might benefit from antibiotic therapy, do Lyme patients reap the reciprocal and benefit from any MS treatments?

RitaA
Posts: 2768
Joined: Thu 1 Jul 2010 8:33

Re: How Inflammation May Impact LNB Patients

Post by RitaA » Tue 1 May 2012 19:46

slmo,

You wrote:
That in mind, if MS patients might benefit from antibiotic therapy, do Lyme patients reap the reciprocal and benefit from any MS treatments?
That's a very good question. I know quite a number of Canadians who were initially diagnosed as having MS before finding out they actually had Lyme disease. Perhaps not surprisingly, those individuals did not respond to the typical medications used to treat MS, but their health did improve after being treated with antibiotics.

According to just one U.S. compounding pharmacy, many thousands of MS patients around the world are finding non-standard treatment with low-dose Naltrexone helpful, and so are at least a handful of Lyme disease patients (myself included). This definitely falls under the category of anecdotal evidence, however, since no large-scale clinical trials have ever been conducted regarding the off-label use of Naltrexone. No pharmaceutical company or government (to date) has been willing to fund that type of research. Naltrexone is a very inexpensive non-narcotic prescription medication, and it must be prepared by a compounding pharmacy for off-label uses (such as MS and pain relief) partly because it is used at about 10% of the normal dosage. Pharmaceutical companies are generally motivated by profits, so they understandably have no interest in promoting the off-label use of an inexpensive medication. Why governments who at least partially subsidize the cost of medications for their citizens aren't funding this type of research is much more difficult for me to understand.

There certainly appears to be some overlap between MS and LNB signs and symptoms in at least a subset of Lyme disease patients. I'm hoping further research will help to shed light on a variety of neurological disorders that seem to have one or more common denominators.

p.s. If you're interested in reading more about the use of low-dose Naltrexone for pain relief, I created this thread back in February:

http://www.lymeneteurope.org/forum/view ... f=7&t=3661

The use of Dexamethasone as an anti-inflammatory (described in the article above) has also been touched upon previously here on LNE. If you do a search using "Dexamethasone", you'll be able to find those older posts.

Edited to add:

Dexamethasone has also been used specifically to suppress immune function in non-human primates, as described here:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC308929/
Infect Immun. 2003 December; 71(12): 7087–7098.

doi: 10.1128/IAI.71.12.7087-7098.2003


PMCID: PMC308929

Infection and Inflammation in Skeletal Muscle from Nonhuman Primates Infected with Different Genospecies of the Lyme Disease Spirochete Borrelia burgdorferi

Diego Cadavid,1,* Yunhong Bai,1 Donna Dail,1 Marie Hurd,1 Kavi Narayan,1 Emir Hodzic,2 Stephen W. Barthold,2 and Andrew R. Pachner1

Department of Neuroscience, University of Medicine and Dentistry of New Jersey—New Jersey Medical School, Newark, New Jersey 07103,1 Center for Comparative Medicine, University of California at Davis, Davis, California 956162

*Corresponding author. Mailing address: UMDNJ—New Jersey Medical School, Department of Neuroscience, 185 South Orange Ave., MSB H506, Newark, NJ 07103

[snip]

All but two NHPs were immunosuppressed with dexamethasone to increase the probability that they would become infected.

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