IAI Accepts, published online ahead of print on 24 February 2014 Infect. Immun. doi:10.1128/IAI.00474-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
Neurons are host cells for Mycobacterium tuberculosis. _________________________________________________________ 1*
Philippa J. Randall, 1*Nai-Jen Hsu, 2Dirk Lang, 2Susan Cooper, 1Boipelo Sebesho, 1Nasiema Allie,
1
Roanne Keeton, 1Ngiambudulu M. Francisco, 1Sumayah Salie, 1Antoinette Labuschagné,3Valerie
Quesniaux, 3Bernhard Ryffel, 2Lauriston Kellaway, 1,4Muazzam Jacobs.
AFFILIATIONS 1
Division of Immunology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty, University of Cape Town, South Africa
2
Department of Human Biology, Health Sciences Faculty, University of Cape Town, South Africa
3
Institut deTransgenose, CNRS, GEM2358, Orleans, France
4
National Health Laboratory Service, South Africa
*Both authors contributed equally to the study.
1
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
ABBREVIATED TITLE M. tuberculosisinfect neurons
KEYWORDS: Neuron, Mycobacterium tuberculosis, infection, microscopy
FUNDING This
study
was
supported
by
the
National
Research
Foundation
(South
Africa),DeutscherAkademischerAustauschDienst, (Germany), the Medical Research Council (South Africa), University of Cape Town and National Health and Laboratory Service (South Africa).
CORRESPONDING AUTHOR Muazzam Jacobs Division of Immunology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty University of Cape Town Observatory, 7925, South Africa. Email: [email protected] Tel: +27 21 406 60 78
ACKNOWLEDGEMENTS We thank Marylin Tyler and Lizette Fick for their sterling contribution to the histopathology. Thank you to Dr Lester Davids and Toni Wiggins for their help in acquiring the SK-N-SH cell-line. A special thank you to Faried Abbass for technical support. We thank the staff of the Division of Immunology and the Animal Unit at UCT for their contribution to animal care and technical support.
2
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
ABSTRACT
1 2 3
Mycobacterium tuberculosis (M. tuberculosis) infection of the central nervous system is thought to
4
be initiated once the bacilli have breached the blood brain barrier and are phagocytosed primarily by
5
microglial cells. In this study the interaction of M. tuberculosis with neurons in vitro and in vivo were
6
investigated. Data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M.
7
tuberculosis bacilli were internalised by murine neuronal cultured cells, in a time dependent manner
8
after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% vs 9.85%).
9
Internalisation of M. Tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical
10
relevance of the findings. Moreover, primary murine hippocampal derived neuronal cultures could
11
similarly internalise M. tuberculosis. Internalised M. tuberculosis bacilli represented a productive
12
infection with retention of bacterial viability and replicative potential, increasing 2-4 fold within 48h.
13
M. tuberculosis bacilli infection of neurons was confirmed in vivo in brains of C57BL/6 mice after
14
intra-cerebral challenge. This study therefore demonstrates neurons as potential new target cells
15
for M. tuberculosis within the central nervous system.
16
3
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
INTRODUCTION
17 18 19
Tuberculosis is primarily a respiratory disease, which is initiated after the inhalation of only a few
20
bacilli and subsequent phagocytosis by alveolar macrophages to establish a local infection focus.
21
Globally, approximately 8.8 million new cases of tuberculosis were reported in 2011, and the
22
disease was associated with 1.45 million deaths (1). Although pulmonary tuberculosis is the
23
predominant form of infection, extra-pulmonary tuberculosis constitutes up to 20% of reported cases
24
of which approximately 1-5% is attributed to tuberculosis of the central nervous system (CNS-TB)
25
(2). CNS-TB occurs primarily in childhood, but significantly increases in adults under conditions of
26
immune suppression, which is associated with considerable morbidity and mortality (3, 4).
27
Pathogenesis of CNS-TB is initiated as a secondary infection during haematogenous dissemination
28
of pulmonary infection to the brain parenchyma (5). Notwithstanding its neuroprotective properties,
29
it has been proposed that M. tuberculosis can cross the blood brain barrier and invade the CNS as
30
free bacilli, supported by studies that illustrated pathogen specific gene upregulation associated with
31
traversal of the blood brain barrier (6). However, the mechanisms associated with evading the
32
protective properties of the blood brain barrier for several bacteria, including M. tuberculosis,
33
remains primarily undefined. Initial events during CNS-TB are characterised by infection of the
34
meninges, the establishment of localised foci and the subsequent release of bacilli into the
35
subarachnoid space (7).
36 37
Several studies have investigated and reported on different cell types that are targeted by M.
38
tuberculosis bacilli for invasion (8, 9, 10, 11). Amongst these macrophages are well described as
39
preferred host cells despite its primary protective function in innate immune responses; the
40
evolutionary development of specific immune evasion mechanisms allowing M. tuberculosis to exist
41
within what is essentially a hostile environment. Studies have also indicated that cells other than
42
macrophages, such as dendritic cells are infected by M. tuberculosis bacilli at a higher rate than was
43
previously thought (11). Differential cytokine profiles produced by infected macrophages and
4
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
44
dendritic cells in comparative studies have suggested that the functional consequences of M.
45
tuberculosis infection of these two distinct cell types may be different (12, 13). Similarly, M.
46
tuberculosis infection of different non-phagocytic cell types may induce responses that are variable.
47
The diversity of cell types that can be infected by M. tuberculosis bacilli, particularly at extra
48
pulmonary sites, suggests that latent infection may be established at such locations. Recent studies
49
demonstrated viable bacilli present in resident macrophages and sinusoidal endothelium cells of the
50
spleen and liver, expressing a genetic profile corresponding to latent infection (14).
51 52
M. tuberculosis bacilli encode specific proteins that actively facilitate entry into cells (15, 16), thereby
53
circumventing the requirement for cells to be phagocytic in order to establish infection. Amongst
54
several intracellular bacterial species that are capable of infecting the central nervous system (17),
55
studies have indicated that microglia are targeted by invading M. tuberculosis bacilli (18, 19),
56
leading to a robust proinflammatory response dependent on NADPH oxidase-dependent reactive
57
oxygen species (ROS) generation (20) and the induction of reactive nitrogen intermediates (21).
58
Neurons have never been shown to be infected by M. tuberculosis bacilli and are not thought be
59
involved in the etiology of the disease. However, neural targeting by M. leprae through binding to
60
laminin-α2 on Schwann cells, has been reported (22) and the presence of M. leprae in the medulla
61
oblongata and spinal cord of patients with lepromatous leprosy was inferred from DNA amplification
62
studies, although the presence of bacilli within neurons was not detected (23). Nonetheless, several
63
pathogenic species do infect neurons including the intracellular bacterium, L. monocytogenes, which
64
can be controlled in an IFNγ dependent bactericidal manner (24, 25).
65 66
In this study the potential of M. tuberculosis bacilli to infect neurons was investigated. Although
67
neurons are generally regarded as non-phagocytic cells, Bowen et al. demonstrated that
68
phagocytosis by different neuronal cell types occur both in vitro and in vivo (26). The phagocytic
69
capability of neurons may therefore largely be unappreciated and under-investigated. Therefore, it
70
was hypothesised thatneuronsare capable of mycobacterial internalisation, thereby affecting
71
neuronal cellular responses. Results obtained in this study conclusively established that M.
5
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
72
tuberculosis bacilli were able to infect neurons directly, as demonstrated by the intracellular location
73
of bacilli through confocal microscopy.
6
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
METHODS
74 75 76
Mice
77
C57BL/6 mice were bred and maintained under specific pathogen-free conditions at the animal
78
facility of the University of Cape Town (South Africa). One day old neonates and adult mice between
79
8-12 weeks of age were used.All animal experimental protocols complied with South African
80
regulations. Approval was obtained from the Animal Research Ethics Committee, University of
81
Cape Town, South Africa (Reference Numbers: AEC 010/017 and AEC 010/018) in accordance with
82
the South African National Standard 10386-The care and use of animals for scientific purposes.
83 84
Mycobacteria
85
Mycobacterium tuberculosis strain H37Rv (Trudeau Mycobacterial Culture Collection, New York),
86
was grown at 37oC in Middlebrook 7H9 broth (Difco™ Laboratories) containing 10% OADC
87
and0.5% Tween-80 until log phase, then aliquoted and stored at -80°C. A frozen aliquot was
88
thawed, passed 30 times through 29 gauge needle and plated in 10 fold serial dilutions on
89
Middlebrook 7H10 agar (Difco™ Laboratories) containing 10% OADC and 0.5% glycerol. The
90
concentration of M. tuberculosis was then determined by counting the colony forming units (CFUs).
91
M. tuberculosis with green fluorescent expressing protein (H37Rv-GFP, provided by Joel Ernst, New
92
York University School of Medicine, USA) was prepared similarly with 25 µg/ml kanamycin added in
93
broth or agar (11).
94 95
Cellcultures and infection
96
Primary microglia were isolated from mixed glial cultures prepared from cerebral cortices of
97
C57BL/6 neonates of either sex, as described previously (27, 28). Cultures of mouse primary
98
neurons were prepared from C57BL/6 day 17 embryos or neonates as described previously with
99
modification (29, 30, 31). Briefly, the dissected hippocampiwere digested, dissociated by trituration,
100
then seeded at a density of 1 X 105cells per well on poly-L-lysinecoated coverslips in 24-well plate
7
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
101
or glass chamber slides. The primary neuronal cultures were maintained in serum-free neurobasal
102
medium (Gibco) containing 2% B27 supplement (Gibco) at 37oC in a 5% CO2 incubator.
103 104
HT22 (murine neuronal cell-line) was obtained from the Salk Institute, California, USA; Neuro-2a
105
(murine neuroblastoma) and BV2 (murine microglia) were obtained from the National Institute for
106
Cancer Research, Genova, Italy. HT22 and Neuro-2a cells were propagated in DMEM with 10%
107
FBS, and BV2 cells were propagated in RPMI 1640 medium with 10% FBS. Human neuroblastoma
108
SK-N-SH (gift from Dr A. Vahidnia, Leiden University Medical Centre, Netherlands) were grown in
109
DMEM supplemented with 10% FBS, and differentiated for 2 weeks in culture with 10 µM of retinoic
110
acid (Sigma) (32).
111 112
To assess optimal cellular infection, neurons were seeded at 1x105 cells/well, while microglia were
113
seeded at 1x104 cells/well, and cultured with M. tuberculosis at a multiplicity of infection (MOI) of
114
2:1, 5:1, 10:1, 30:1, 50:1 or 100:1 (M. tuberculosis : cells). Although association of M. tuberculosis
115
with cultured neuronal cells occurred at all ratios (data not shown), an MOI of 30:1 was deemed
116
optimal, supported by other studies usingan ex-vivo model of hippocampal neuronal infection with L.
117
monocytogenes (25); while an MOI of 2:1 was found optimal for microglial infection. Thus briefly,
118
HT22, Neuro2a and primary hippocampal neurons were infected with H37Rv-GFP at an MOI of 30:1
119
and microglia at an MOI of 2:1 for 4h in 24 well culture plates, after which culture medium was
120
replaced with fresh medium containing 50 µg/ml Gentamicin (Sigma). For image studies, cells were
121
fixed for stainingat 6h, 24h and 48h post infection for further analysis. For colony enumeration,
122
neurons were extensively washed with PBS, lysed with 1% TritonX100 in PBS and lysates cultured
123
on Middlebrook 7H10 agar (Difco) supplemented with 10%OADC, 0.5% glycerol and 25 µg/ml
124
kanamycin. Mycobacterial cultures were incubated inside semi-sealed plastic bags at 37°C for 18-
125
21 days and colony counts registered.
126 127
Histology and immunocytochemistry of infected cultures
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M. tuberculosis infect neurons
Randall and Hsu et al, 2013
128
For histology, H37Rv infected cells were fixed with 4% paraformaldehyde (PFA) in PBS, washed
129
and subjected to Ziehl-Neelsen (ZN) staining for acid fast bacilli. For immunohistochemical analysis,
130
H37Rv-GFP infected cells were fixed with 4% PFA, washed and blocked with 1% BSA, then
131
incubated with either alexa 555-conjugated phalloidin (1:600; Invitrogen) or anti-microtubule-
132
associated protein-2 (MAP-2)(1:1000, Abcam) and visualised with Cy3-conjugated secondary
133
antibody (1:1000, Jackson ImmunoResearch Laboratories). SK-N-SH cell nuclei were stained with
134
6-diamidino-phenylidone (DAPI; Sigma). The immunoflourescent labelled cultures were mounted in
135
fluorescent mounting medium (Dako) and images captured with Zeiss LSM 510 confocal
136
microscope (Oberkochen, Germany). High-resolution confocal imaging was carried out using a 63x
137
(1.4NA) oil immersion objective and z-stacks acquired using Nyqvist sampling parameters.
138 139
The quantification of M. tuberculosis associated cultured cells was performed using light microscopy,
140
where 100-400 microglial or neuronal cells were examined under bright field for each experiment.
141
Cultured neurons or microglia, which associated with M. tuberculosis bacilli was calculated as the
142
number of cells associated with acid fast bacilli out of the total number of cells examined.
143 144
The quantification of cells, which contained internalised M. tuberculosis bacilli was performed using
145
confocal microscopy where 100-150 cells per cell-type were examined for each experiment. The
146
cells containing internalised bacilli were counted in the 3-dimentional z-stack images of 5 different
147
fields per cell-type. The percentage of internalisation was calculated as the number of cells
148
containing intracellular H37Rv-GFP bacilli out of the total number of cells counted in the z-stack
149
images.
150 151
Intra-cerebral infection and immunohistochemistry of infected mouse
152
Six of C57BL/6 female mice (8-12 weeks) from 2 independent experiments were inoculated intra-
153
cerebrally in the left cerebral hemisphere with 1X105 CFU of H37Rv-GFP using Hamilton syringe
154
(Gastight no.1701, Switzerland). At 7 and 14 days post infection, infected mice were deeply
155
anaesthetised and transcardially perfused with 4% PFA. The 40µm brain sections were cut using
9
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
156
vibratome then processed for immunohistochemistry. The microglia and astrocytes were labelled
157
with CD11b (Clone: [M1/70], 1:500; Abcam) and glial-fibrillary acidic protein antibody (GFAP; 1:1000;
158
Sigma) respectively. The neurons were labelled with either NeuN (Clone: A60, 1:100, Merck-
159
Millipore) or MAP2 (1:1000, Abcam) or β-III-tubulin antibody (1:1000; Abcam), and corresponding
160
Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The nuclei were
161
stained with DAPI (Sigma). The immunofluorescentlabelled sections were mounted in fluorescent
162
mounting medium and viewed with Zeiss 510LSM unit.
163 164
Lysotracker staining
165
To effectively label and track phagolysosome in live cells, infected cultures were incubated with
166
Lysotracker® (Molecular Probes, USA) before fixation and permeablization. Two hours prior to
167
experimental time points, Lysotracker® (1:900) was added to the culture supernatant at 37°C. At
168
24h or 48h post infection, the cells were fixed with 4% PFA in PBS, washed and blocked with 1%
169
BSA in PBS. To ensure sufficient staining, the cells were incubated again with Lysotracker® (1:1000)
170
overnight at 4°C. Subsequently, nuclei were stained with DAPI, and the coverslips were mounted in
171
fluorescent mounting medium (Dako, CA, USA).
172 173
Cytokine quantification
174
Supernatants from neuronal cultures were frozen at -80ºC until further analysis. Cytokine
175
concentrations were determined by ELISA using reagents from R&D Systems (Minneapolis, MN)
176
according to the instructions of the manufacturers.
177 178
Statistical analysis
179
The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed
180
by one way ANOVA and one-tailed t-test for comparisons among the time points. For all tests, a p-
181
value of < 0.05 was considered significant.
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M. tuberculosis infect neurons
Randall and Hsu et al, 2013
RESULTS
182 183 184
M. tuberculosis bacilli associate with neuronal cultures.
185
In initial studies the microglial cell line, BV2 and cortex derived primary microglia were cultured in
186
the presence of M. tuberculosis bacilli at a MOI of 2:1 for 6h, 24h and 48h as established phagocytic
187
cells against which to measure neuronal-bacilli interaction. A time dependent association of M.
188
tuberculosis bacilli was observed in both BV2 (Fig.1A and Fig.1B) and primary microglial cultures
189
(Fig.1C and Fig.1D) with >30% host cells representing microglia-bacilli interaction. Next, neuronal
190
cultures of immortalised HT22 cells (Fig.2A and Fig.2B) and Neuro-2a cells (Fig.2C and Fig.2D)
191
were exposed to M. tuberculosis bacilli at an MOI of 30:1 for 6h,24h and 48h. Analysis of infected
192
neuronal cultures showed that M. tuberculosis bacilli were associated with the soma and the
193
neurites. Quantitatively there was a time dependent increase in association of M. tuberculosis bacilli
194
with both neuronal cell lines, and anoverall higher number of HT22 cells associated with bacilli
195
compared to Neuro-2a cells at all time points investigated (Table 1). Although Neuro-2a cells and
196
HT22 cells are widely used to characterise neuronal behaviour and responses, in general,
197
transformed cell lines may differ from primary cells with respect to structure, development, binding
198
and functional characteristics (33). Therefore, to confirm the neuronal infectivity of M. tuberculosis
199
bacilli, and to exclude that previously observed mycobacterial association was due to anomalous
200
characteristics of neuronal cell lines, the observation was validated in primary neuronal cell cultures.
201
Primary neuronal cell cultures were established from murine hippocampi and infected with M.
202
tuberculosis bacilli at an MOI of 30:1 for 6h, 24h and 48h. Uptake in primary neuronal cultures was
203
comparable to HT22 cells and Neuro-2a cells; M. tuberculosis bacilli displayed a time dependent
204
increase in association from 7.9% to 21.3% over a 48h period (Table 1) with bacilli present
205
associated with the neurites and cell bodies (Fig.2E and Fig.2F). Therefore, for the first time, the
206
dataclearly demonstrates that M. tuberculosis is capable of direct interaction with neurons.
207 208
Murine neurons internalise M. tuberculosis bacilli in vitro.
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M. tuberculosis infect neurons
Randall and Hsu et al, 2013
209
Observations of an association of bacilli with cultured neurons after Ziehl-Neelsen staining may
210
have represented bound, extracellular bacilli rather than bacteria that were intracellular. To
211
investigate whether M. tuberculosis bacilli were indeed internalised by neurons, the neuronal cell
212
lines HT22 (Fig.3A-C) and Neuro-2a (Fig.3D-F), as well as primary neuronal cultures (Fig3.G-I)
213
were infected with a recombinant GFP expressingM. tuberculosis H37Rv strain at an MOI of 30:1.
214
Similarly, BV2 microglial cells (Fig.3J-L) were infected at an MOI of 2:1 to act as a positive control
215
cell strain for bacterial internalisation. To confirm cytoplasmic localisation of bacilli in microglia,
216
cultures were stained with cytoflourochrome-conjugated phalloidin, a cytoskeletal marker and
217
analysed by confocal microscopy. Analysis of Z-stack images showed that in most cases multiple
218
bacilli were phagocytosed by BV2 (Fig.3K) and orthogonal views confirmed that all bacilli were
219
internalised (Fig.3L). To determine whether M. tuberculosis bacilli were internalised by neurons,
220
infected HT22 and Neuro-2a cultures were stained with cytoflourochrome-conjugated phalloidin
221
while infected primary neurons were identified with the neuronal specific marker MAP2. The
222
orthogonal display of the three dimensional data demonstrated internalised M. tuberculosis bacilli in
223
HT22 (Fig.3C), Neuro-2a (Fig.3F) and primary neuronal cultures (Fig.3I), embedded within
224
cytoplasmic structures, as identified by the different markers.
225 226
Quantificationof the number of cells with bacilli showed ≥98% of microglial cell associated bacilli
227
were internalised after 24h and 48h (Table 1). Importantly, for HT22, 11.8% and 17.7% of cultured
228
cells internalisedM. Tuberculosis bacilli after 24h and 48h respectively (Table 1). Although Neuro-
229
2a internalisation was approximately 2X lower compared to HT22, significant internalisation levels of
230
6.2% and 9.8% at 24h and 48h were nonetheless measured (Table 1).
231
cultures, 11.2% of cells had internalised bacilli at 24h,which decreased slightly to 10.1% after 48h
232
(Table 1). Overall, relative ratios indicated that, whereas microglial cells internalised most of the
233
associated bacilli, murine neuronal cells only internalise 45-65% of associated bacilli after 48h
234
(Table 1). Moreover, to determine whether internalised M. tuberculosiswithin neurons represent a
235
productive infection, where bacilli remain viable and retain the capacity to replicate, infected HT22,
236
Neuro-2a and primary neurons were lysed at 6h, 24h and 48h and bacterial colonies enumerated.
For primary neuronal
12
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
237
The data indicated that initial uptake and internalisation of mycobacteria within primary neurons may
238
be superior to HT22 and Neuro-2ahaving significantly higher bacilli levels at 6h (Fig 3M). Notably,
239
bacilli growth occurred in all neuron cultures with significantly higher numbers recorded at 48h
240
compared to 6h (Fig. 3M).
241 242
Thus, the study provided evidence that neurons are capable of internalising M. tuberculosis bacilli,
243
which is viable and capable of replication within neurons.
244 245
Neurons internalise M. tuberculosis bacilli in vivo
246
In this study, the localisation of GFP expressing M. tuberculosis (green) in relation to two different
247
intracellular neuronal specific markers, β-III-tubulin (red) and MAP2 (red), were analysed and results
248
compared to microglial uptake. Orthogonal projections (Fig.4B, D and F) representing 3-dimensional
249
data sets provide a view of the x-y as well as the z-dimension of the original z-stack.Cell nuclei are
250
labelled with DAPI. Respective differential interference contract (DIC) images (Fig.4A, C and E),
251
illustrate the position of M. tuberculosis-GFP within the tissue. From the data presented in Fig.4B it
252
was clear that M. tuberculosis-GFP bacilli were contained within β-III-tubulin-positive neuronal
253
structures (red), indicated by partial co-localisation (yellow) of the red and green fluorescence
254
signals. Similarly, bacilli resided within MAP2 positive neuronal structures (red), which resulted in a
255
yellow co-localisation signal (Fig.4D). Green fluorescent bacilli were also present although it was
256
unclear whether the latter represented free or internalised bacilli where MAP2 did not condense
257
around the bacillus; limitation in optical resolution preventing the delineation of cellular structure.
258
Microscopic observations indicated that CD11b+ microglial cells contained higher numbers of bacilli
259
compared to neurons. CD11b as a cell surface antigen allows for sufficient spatial separation of the
260
red signal on the surface and the green signal from the internalised bacilli not to result in a co-
261
localised (yellow) signal clearly evident in Fig.4F.
262 263
Therefore the data demonstrates that M. tuberculosis bacilli can be internalised by neurons both
264
under in vitro and in vivo conditions, but that neuronal uptake is subservient to that of microglia.
13
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
265 266
M. tuberculosisis bacilli encapsulated within neuronal cytoskeletal structures.
267
To gain additional insight into the relationship between neuronal structures and internalised bacilli,
268
localised cytoplasmic sites were further investigatedin vitro and in vivo. Bacilli may display distinct
269
condensation of cytoskeletal elements (MAP2+) around it, which resulted in partial co-localisation of
270
the fluorescent signals (yellow), but still allowed for separation of the structures in vitro (Fig.5A).
271
Fig.5B confirmed neuronal internalisation of the bacillus, whereas closer inspection and analysis of
272
detailed images (Fig.5B1-B3) revealed that the bacillus resided within a MAP2-positive capsule-like
273
cytoplasmic neuronal structure indicative of rearrangement of the cytoskeleton during infection.
274
Similarly, this co-localisation was observed in vivo. As described above, in Fig.4B and Fig.4D, co-
275
localisation indicated M. tuberculosisinfection of neurons within the CNS following intra-cerebral
276
challenge. Scan resolution of these sections, due to background and tissue thickness (40µm), was
277
not sufficient to separate the red and green immunofluorescent signals from cytoplasmic markers
278
and bacilli, respectively. However, the MAP2 panel (Fig.5D) showed MAP2 condensation within the
279
area surrounding the bacillus. Therefore, it is plausible that thein vivo infection of neurons by M
280
tuberculosis (Fig.5C-E) likely reflects conditions as observed in vitro (Fig.5B1-B3).
281 282
Cultured human neuronal cells internalise M. tuberculosis bacilli.
283
The data demonstrated M. tuberculosis internalisation by murine derived neuronal cell lines and
284
primary murine neuronal cultures, which were confirmed by neuronal uptakein vivo. To ascertain
285
whether these observations have clinical relevance, we infected retinoic acid differentiated SK-N-SH
286
human derived neuroblastoma cell cultures with M. tuberculosis-GFP bacilli at an MOI of 30:1. The
287
cytoplasmic localisation of fluorescent bacilli within SK-N-SH cultures was confirmed through
288
staining with the neuronal specific markers β-III-tubulin or MAP2. Similar to observations described
289
for murine derived neuronal cultures, the orthogonal display of the three dimensional data
290
demonstrated internalised M. tuberculosis bacilli in either β-III-tubulin positive (Fig.6C) or MAP2
291
positive (Fig.6F) cytoplasmic structures. In addition, internalisation was confirmed via the co-
292
localisation of the green (bacilli) and red (β-III-tubulin or MAP2) fluorescence signals resulting in a
14
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
293
partially yellow fluorescent signal (Fig.6C and 6F). Quantification of the percentage of SK-N-SH cells
294
associated with bacilli showed a statistically significant increase from 10.2% to 21% over 48h (Table
295
1), similar to observations in murine derived neuronal cultures. The percentage of cells, which
296
internalised bacilli increased from 11.9% to 18.5% from 24h to 48h (Table 1). This study therefore
297
confirms that human neuronal cells are capable of being infected by M. tuberculosis bacilli.
298 299
Limited association of M. tuberculosis bacilli with phagolysosomes during neuronal infection
300
Previous studies have demonstrated that neurons are capable of phagocytosis (26) thereby
301
introducing the possibility that M. tuberculosis bacilli may be internalised in this manner. We
302
investigated M. tuberculosis bacilli association with phagolysosomes in primary neuronal cultures
303
infected at a MOI of 30:1 for 24h and 48h, respectively. Analysis of confocal images showed
304
predominant separation of the Lysotracker Red marker and green fluorescent bacilli, which indicated
305
either arrest of phagosome-lysosomal fusion during neuron infection or alternatively, residence of
306
bacilli within the cytosol and not within phagosomes (Fig.7).
307 308
Neurons are activated during M. tuberculosis infection
309
Neurons are capable of generating an immune response when confronted by various pathogens.
310
However, neuronal activation in response to M. tuberculosis bacilli has not yet been reported. In
311
this study IL-1β, IL-6 and IL-10 cytokine expression was measured in M. tuberculosis bacilli infected
312
HT22, Neuro-2a and primary neuronsas an indication of functional response (Figure 8). Both
313
neuronal cell lines and primary neuronal cultures had significantly higher (p
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
Neurons are host cells for Mycobacterium tuberculosis. _________________________________________________________ 1*
Philippa J. Randall, 1*Nai-Jen Hsu, 2Dirk Lang, 2Susan Cooper, 1Boipelo Sebesho, 1Nasiema Allie,
1
Roanne Keeton, 1Ngiambudulu M. Francisco, 1Sumayah Salie, 1Antoinette Labuschagné,3Valerie
Quesniaux, 3Bernhard Ryffel, 2Lauriston Kellaway, 1,4Muazzam Jacobs.
AFFILIATIONS 1
Division of Immunology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty, University of Cape Town, South Africa
2
Department of Human Biology, Health Sciences Faculty, University of Cape Town, South Africa
3
Institut deTransgenose, CNRS, GEM2358, Orleans, France
4
National Health Laboratory Service, South Africa
*Both authors contributed equally to the study.
1
M. tuberculosis infect neurons
Randall and Hsu et al, 2013
ABBREVIATED TITLE M. tuberculosisinfect neurons
KEYWORDS: Neuron, Mycobacterium tuberculosis, infection, microscopy
FUNDING This
study
was
supported
by
the
National
Research
Foundation
(South
Africa),DeutscherAkademischerAustauschDienst, (Germany), the Medical Research Council (South Africa), University of Cape Town and National Health and Laboratory Service (South Africa).
CORRESPONDING AUTHOR Muazzam Jacobs Division of Immunology, Institute of Infectious Disease and Molecular Medicine, Health Sciences Faculty University of Cape Town Observatory, 7925, South Africa. Email: [email protected] Tel: +27 21 406 60 78
ACKNOWLEDGEMENTS We thank Marylin Tyler and Lizette Fick for their sterling contribution to the histopathology. Thank you to Dr Lester Davids and Toni Wiggins for their help in acquiring the SK-N-SH cell-line. A special thank you to Faried Abbass for technical support. We thank the staff of the Division of Immunology and the Animal Unit at UCT for their contribution to animal care and technical support.
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ABSTRACT
1 2 3
Mycobacterium tuberculosis (M. tuberculosis) infection of the central nervous system is thought to
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be initiated once the bacilli have breached the blood brain barrier and are phagocytosed primarily by
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microglial cells. In this study the interaction of M. tuberculosis with neurons in vitro and in vivo were
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investigated. Data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M.
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tuberculosis bacilli were internalised by murine neuronal cultured cells, in a time dependent manner
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after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% vs 9.85%).
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Internalisation of M. Tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical
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relevance of the findings. Moreover, primary murine hippocampal derived neuronal cultures could
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similarly internalise M. tuberculosis. Internalised M. tuberculosis bacilli represented a productive
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infection with retention of bacterial viability and replicative potential, increasing 2-4 fold within 48h.
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M. tuberculosis bacilli infection of neurons was confirmed in vivo in brains of C57BL/6 mice after
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intra-cerebral challenge. This study therefore demonstrates neurons as potential new target cells
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for M. tuberculosis within the central nervous system.
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INTRODUCTION
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Tuberculosis is primarily a respiratory disease, which is initiated after the inhalation of only a few
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bacilli and subsequent phagocytosis by alveolar macrophages to establish a local infection focus.
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Globally, approximately 8.8 million new cases of tuberculosis were reported in 2011, and the
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disease was associated with 1.45 million deaths (1). Although pulmonary tuberculosis is the
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predominant form of infection, extra-pulmonary tuberculosis constitutes up to 20% of reported cases
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of which approximately 1-5% is attributed to tuberculosis of the central nervous system (CNS-TB)
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(2). CNS-TB occurs primarily in childhood, but significantly increases in adults under conditions of
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immune suppression, which is associated with considerable morbidity and mortality (3, 4).
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Pathogenesis of CNS-TB is initiated as a secondary infection during haematogenous dissemination
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of pulmonary infection to the brain parenchyma (5). Notwithstanding its neuroprotective properties,
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it has been proposed that M. tuberculosis can cross the blood brain barrier and invade the CNS as
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free bacilli, supported by studies that illustrated pathogen specific gene upregulation associated with
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traversal of the blood brain barrier (6). However, the mechanisms associated with evading the
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protective properties of the blood brain barrier for several bacteria, including M. tuberculosis,
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remains primarily undefined. Initial events during CNS-TB are characterised by infection of the
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meninges, the establishment of localised foci and the subsequent release of bacilli into the
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subarachnoid space (7).
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Several studies have investigated and reported on different cell types that are targeted by M.
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tuberculosis bacilli for invasion (8, 9, 10, 11). Amongst these macrophages are well described as
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preferred host cells despite its primary protective function in innate immune responses; the
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evolutionary development of specific immune evasion mechanisms allowing M. tuberculosis to exist
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within what is essentially a hostile environment. Studies have also indicated that cells other than
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macrophages, such as dendritic cells are infected by M. tuberculosis bacilli at a higher rate than was
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previously thought (11). Differential cytokine profiles produced by infected macrophages and
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dendritic cells in comparative studies have suggested that the functional consequences of M.
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tuberculosis infection of these two distinct cell types may be different (12, 13). Similarly, M.
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tuberculosis infection of different non-phagocytic cell types may induce responses that are variable.
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The diversity of cell types that can be infected by M. tuberculosis bacilli, particularly at extra
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pulmonary sites, suggests that latent infection may be established at such locations. Recent studies
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demonstrated viable bacilli present in resident macrophages and sinusoidal endothelium cells of the
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spleen and liver, expressing a genetic profile corresponding to latent infection (14).
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M. tuberculosis bacilli encode specific proteins that actively facilitate entry into cells (15, 16), thereby
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circumventing the requirement for cells to be phagocytic in order to establish infection. Amongst
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several intracellular bacterial species that are capable of infecting the central nervous system (17),
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studies have indicated that microglia are targeted by invading M. tuberculosis bacilli (18, 19),
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leading to a robust proinflammatory response dependent on NADPH oxidase-dependent reactive
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oxygen species (ROS) generation (20) and the induction of reactive nitrogen intermediates (21).
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Neurons have never been shown to be infected by M. tuberculosis bacilli and are not thought be
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involved in the etiology of the disease. However, neural targeting by M. leprae through binding to
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laminin-α2 on Schwann cells, has been reported (22) and the presence of M. leprae in the medulla
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oblongata and spinal cord of patients with lepromatous leprosy was inferred from DNA amplification
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studies, although the presence of bacilli within neurons was not detected (23). Nonetheless, several
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pathogenic species do infect neurons including the intracellular bacterium, L. monocytogenes, which
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can be controlled in an IFNγ dependent bactericidal manner (24, 25).
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In this study the potential of M. tuberculosis bacilli to infect neurons was investigated. Although
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neurons are generally regarded as non-phagocytic cells, Bowen et al. demonstrated that
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phagocytosis by different neuronal cell types occur both in vitro and in vivo (26). The phagocytic
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capability of neurons may therefore largely be unappreciated and under-investigated. Therefore, it
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was hypothesised thatneuronsare capable of mycobacterial internalisation, thereby affecting
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neuronal cellular responses. Results obtained in this study conclusively established that M.
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tuberculosis bacilli were able to infect neurons directly, as demonstrated by the intracellular location
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of bacilli through confocal microscopy.
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METHODS
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Mice
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C57BL/6 mice were bred and maintained under specific pathogen-free conditions at the animal
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facility of the University of Cape Town (South Africa). One day old neonates and adult mice between
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8-12 weeks of age were used.All animal experimental protocols complied with South African
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regulations. Approval was obtained from the Animal Research Ethics Committee, University of
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Cape Town, South Africa (Reference Numbers: AEC 010/017 and AEC 010/018) in accordance with
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the South African National Standard 10386-The care and use of animals for scientific purposes.
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Mycobacteria
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Mycobacterium tuberculosis strain H37Rv (Trudeau Mycobacterial Culture Collection, New York),
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was grown at 37oC in Middlebrook 7H9 broth (Difco™ Laboratories) containing 10% OADC
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and0.5% Tween-80 until log phase, then aliquoted and stored at -80°C. A frozen aliquot was
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thawed, passed 30 times through 29 gauge needle and plated in 10 fold serial dilutions on
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Middlebrook 7H10 agar (Difco™ Laboratories) containing 10% OADC and 0.5% glycerol. The
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concentration of M. tuberculosis was then determined by counting the colony forming units (CFUs).
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M. tuberculosis with green fluorescent expressing protein (H37Rv-GFP, provided by Joel Ernst, New
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York University School of Medicine, USA) was prepared similarly with 25 µg/ml kanamycin added in
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broth or agar (11).
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Cellcultures and infection
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Primary microglia were isolated from mixed glial cultures prepared from cerebral cortices of
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C57BL/6 neonates of either sex, as described previously (27, 28). Cultures of mouse primary
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neurons were prepared from C57BL/6 day 17 embryos or neonates as described previously with
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modification (29, 30, 31). Briefly, the dissected hippocampiwere digested, dissociated by trituration,
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then seeded at a density of 1 X 105cells per well on poly-L-lysinecoated coverslips in 24-well plate
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or glass chamber slides. The primary neuronal cultures were maintained in serum-free neurobasal
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medium (Gibco) containing 2% B27 supplement (Gibco) at 37oC in a 5% CO2 incubator.
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HT22 (murine neuronal cell-line) was obtained from the Salk Institute, California, USA; Neuro-2a
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(murine neuroblastoma) and BV2 (murine microglia) were obtained from the National Institute for
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Cancer Research, Genova, Italy. HT22 and Neuro-2a cells were propagated in DMEM with 10%
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FBS, and BV2 cells were propagated in RPMI 1640 medium with 10% FBS. Human neuroblastoma
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SK-N-SH (gift from Dr A. Vahidnia, Leiden University Medical Centre, Netherlands) were grown in
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DMEM supplemented with 10% FBS, and differentiated for 2 weeks in culture with 10 µM of retinoic
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acid (Sigma) (32).
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To assess optimal cellular infection, neurons were seeded at 1x105 cells/well, while microglia were
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seeded at 1x104 cells/well, and cultured with M. tuberculosis at a multiplicity of infection (MOI) of
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2:1, 5:1, 10:1, 30:1, 50:1 or 100:1 (M. tuberculosis : cells). Although association of M. tuberculosis
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with cultured neuronal cells occurred at all ratios (data not shown), an MOI of 30:1 was deemed
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optimal, supported by other studies usingan ex-vivo model of hippocampal neuronal infection with L.
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monocytogenes (25); while an MOI of 2:1 was found optimal for microglial infection. Thus briefly,
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HT22, Neuro2a and primary hippocampal neurons were infected with H37Rv-GFP at an MOI of 30:1
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and microglia at an MOI of 2:1 for 4h in 24 well culture plates, after which culture medium was
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replaced with fresh medium containing 50 µg/ml Gentamicin (Sigma). For image studies, cells were
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fixed for stainingat 6h, 24h and 48h post infection for further analysis. For colony enumeration,
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neurons were extensively washed with PBS, lysed with 1% TritonX100 in PBS and lysates cultured
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on Middlebrook 7H10 agar (Difco) supplemented with 10%OADC, 0.5% glycerol and 25 µg/ml
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kanamycin. Mycobacterial cultures were incubated inside semi-sealed plastic bags at 37°C for 18-
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21 days and colony counts registered.
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Histology and immunocytochemistry of infected cultures
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For histology, H37Rv infected cells were fixed with 4% paraformaldehyde (PFA) in PBS, washed
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and subjected to Ziehl-Neelsen (ZN) staining for acid fast bacilli. For immunohistochemical analysis,
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H37Rv-GFP infected cells were fixed with 4% PFA, washed and blocked with 1% BSA, then
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incubated with either alexa 555-conjugated phalloidin (1:600; Invitrogen) or anti-microtubule-
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associated protein-2 (MAP-2)(1:1000, Abcam) and visualised with Cy3-conjugated secondary
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antibody (1:1000, Jackson ImmunoResearch Laboratories). SK-N-SH cell nuclei were stained with
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6-diamidino-phenylidone (DAPI; Sigma). The immunoflourescent labelled cultures were mounted in
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fluorescent mounting medium (Dako) and images captured with Zeiss LSM 510 confocal
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microscope (Oberkochen, Germany). High-resolution confocal imaging was carried out using a 63x
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(1.4NA) oil immersion objective and z-stacks acquired using Nyqvist sampling parameters.
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The quantification of M. tuberculosis associated cultured cells was performed using light microscopy,
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where 100-400 microglial or neuronal cells were examined under bright field for each experiment.
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Cultured neurons or microglia, which associated with M. tuberculosis bacilli was calculated as the
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number of cells associated with acid fast bacilli out of the total number of cells examined.
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The quantification of cells, which contained internalised M. tuberculosis bacilli was performed using
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confocal microscopy where 100-150 cells per cell-type were examined for each experiment. The
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cells containing internalised bacilli were counted in the 3-dimentional z-stack images of 5 different
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fields per cell-type. The percentage of internalisation was calculated as the number of cells
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containing intracellular H37Rv-GFP bacilli out of the total number of cells counted in the z-stack
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images.
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Intra-cerebral infection and immunohistochemistry of infected mouse
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Six of C57BL/6 female mice (8-12 weeks) from 2 independent experiments were inoculated intra-
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cerebrally in the left cerebral hemisphere with 1X105 CFU of H37Rv-GFP using Hamilton syringe
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(Gastight no.1701, Switzerland). At 7 and 14 days post infection, infected mice were deeply
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anaesthetised and transcardially perfused with 4% PFA. The 40µm brain sections were cut using
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vibratome then processed for immunohistochemistry. The microglia and astrocytes were labelled
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with CD11b (Clone: [M1/70], 1:500; Abcam) and glial-fibrillary acidic protein antibody (GFAP; 1:1000;
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Sigma) respectively. The neurons were labelled with either NeuN (Clone: A60, 1:100, Merck-
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Millipore) or MAP2 (1:1000, Abcam) or β-III-tubulin antibody (1:1000; Abcam), and corresponding
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Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The nuclei were
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stained with DAPI (Sigma). The immunofluorescentlabelled sections were mounted in fluorescent
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mounting medium and viewed with Zeiss 510LSM unit.
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Lysotracker staining
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To effectively label and track phagolysosome in live cells, infected cultures were incubated with
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Lysotracker® (Molecular Probes, USA) before fixation and permeablization. Two hours prior to
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experimental time points, Lysotracker® (1:900) was added to the culture supernatant at 37°C. At
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24h or 48h post infection, the cells were fixed with 4% PFA in PBS, washed and blocked with 1%
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BSA in PBS. To ensure sufficient staining, the cells were incubated again with Lysotracker® (1:1000)
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overnight at 4°C. Subsequently, nuclei were stained with DAPI, and the coverslips were mounted in
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fluorescent mounting medium (Dako, CA, USA).
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Cytokine quantification
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Supernatants from neuronal cultures were frozen at -80ºC until further analysis. Cytokine
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concentrations were determined by ELISA using reagents from R&D Systems (Minneapolis, MN)
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according to the instructions of the manufacturers.
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Statistical analysis
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The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed
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by one way ANOVA and one-tailed t-test for comparisons among the time points. For all tests, a p-
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value of < 0.05 was considered significant.
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RESULTS
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M. tuberculosis bacilli associate with neuronal cultures.
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In initial studies the microglial cell line, BV2 and cortex derived primary microglia were cultured in
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the presence of M. tuberculosis bacilli at a MOI of 2:1 for 6h, 24h and 48h as established phagocytic
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cells against which to measure neuronal-bacilli interaction. A time dependent association of M.
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tuberculosis bacilli was observed in both BV2 (Fig.1A and Fig.1B) and primary microglial cultures
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(Fig.1C and Fig.1D) with >30% host cells representing microglia-bacilli interaction. Next, neuronal
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cultures of immortalised HT22 cells (Fig.2A and Fig.2B) and Neuro-2a cells (Fig.2C and Fig.2D)
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were exposed to M. tuberculosis bacilli at an MOI of 30:1 for 6h,24h and 48h. Analysis of infected
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neuronal cultures showed that M. tuberculosis bacilli were associated with the soma and the
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neurites. Quantitatively there was a time dependent increase in association of M. tuberculosis bacilli
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with both neuronal cell lines, and anoverall higher number of HT22 cells associated with bacilli
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compared to Neuro-2a cells at all time points investigated (Table 1). Although Neuro-2a cells and
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HT22 cells are widely used to characterise neuronal behaviour and responses, in general,
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transformed cell lines may differ from primary cells with respect to structure, development, binding
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and functional characteristics (33). Therefore, to confirm the neuronal infectivity of M. tuberculosis
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bacilli, and to exclude that previously observed mycobacterial association was due to anomalous
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characteristics of neuronal cell lines, the observation was validated in primary neuronal cell cultures.
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Primary neuronal cell cultures were established from murine hippocampi and infected with M.
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tuberculosis bacilli at an MOI of 30:1 for 6h, 24h and 48h. Uptake in primary neuronal cultures was
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comparable to HT22 cells and Neuro-2a cells; M. tuberculosis bacilli displayed a time dependent
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increase in association from 7.9% to 21.3% over a 48h period (Table 1) with bacilli present
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associated with the neurites and cell bodies (Fig.2E and Fig.2F). Therefore, for the first time, the
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dataclearly demonstrates that M. tuberculosis is capable of direct interaction with neurons.
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Murine neurons internalise M. tuberculosis bacilli in vitro.
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Observations of an association of bacilli with cultured neurons after Ziehl-Neelsen staining may
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have represented bound, extracellular bacilli rather than bacteria that were intracellular. To
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investigate whether M. tuberculosis bacilli were indeed internalised by neurons, the neuronal cell
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lines HT22 (Fig.3A-C) and Neuro-2a (Fig.3D-F), as well as primary neuronal cultures (Fig3.G-I)
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were infected with a recombinant GFP expressingM. tuberculosis H37Rv strain at an MOI of 30:1.
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Similarly, BV2 microglial cells (Fig.3J-L) were infected at an MOI of 2:1 to act as a positive control
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cell strain for bacterial internalisation. To confirm cytoplasmic localisation of bacilli in microglia,
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cultures were stained with cytoflourochrome-conjugated phalloidin, a cytoskeletal marker and
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analysed by confocal microscopy. Analysis of Z-stack images showed that in most cases multiple
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bacilli were phagocytosed by BV2 (Fig.3K) and orthogonal views confirmed that all bacilli were
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internalised (Fig.3L). To determine whether M. tuberculosis bacilli were internalised by neurons,
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infected HT22 and Neuro-2a cultures were stained with cytoflourochrome-conjugated phalloidin
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while infected primary neurons were identified with the neuronal specific marker MAP2. The
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orthogonal display of the three dimensional data demonstrated internalised M. tuberculosis bacilli in
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HT22 (Fig.3C), Neuro-2a (Fig.3F) and primary neuronal cultures (Fig.3I), embedded within
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cytoplasmic structures, as identified by the different markers.
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Quantificationof the number of cells with bacilli showed ≥98% of microglial cell associated bacilli
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were internalised after 24h and 48h (Table 1). Importantly, for HT22, 11.8% and 17.7% of cultured
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cells internalisedM. Tuberculosis bacilli after 24h and 48h respectively (Table 1). Although Neuro-
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2a internalisation was approximately 2X lower compared to HT22, significant internalisation levels of
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6.2% and 9.8% at 24h and 48h were nonetheless measured (Table 1).
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cultures, 11.2% of cells had internalised bacilli at 24h,which decreased slightly to 10.1% after 48h
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(Table 1). Overall, relative ratios indicated that, whereas microglial cells internalised most of the
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associated bacilli, murine neuronal cells only internalise 45-65% of associated bacilli after 48h
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(Table 1). Moreover, to determine whether internalised M. tuberculosiswithin neurons represent a
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productive infection, where bacilli remain viable and retain the capacity to replicate, infected HT22,
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Neuro-2a and primary neurons were lysed at 6h, 24h and 48h and bacterial colonies enumerated.
For primary neuronal
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The data indicated that initial uptake and internalisation of mycobacteria within primary neurons may
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be superior to HT22 and Neuro-2ahaving significantly higher bacilli levels at 6h (Fig 3M). Notably,
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bacilli growth occurred in all neuron cultures with significantly higher numbers recorded at 48h
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compared to 6h (Fig. 3M).
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Thus, the study provided evidence that neurons are capable of internalising M. tuberculosis bacilli,
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which is viable and capable of replication within neurons.
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Neurons internalise M. tuberculosis bacilli in vivo
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In this study, the localisation of GFP expressing M. tuberculosis (green) in relation to two different
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intracellular neuronal specific markers, β-III-tubulin (red) and MAP2 (red), were analysed and results
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compared to microglial uptake. Orthogonal projections (Fig.4B, D and F) representing 3-dimensional
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data sets provide a view of the x-y as well as the z-dimension of the original z-stack.Cell nuclei are
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labelled with DAPI. Respective differential interference contract (DIC) images (Fig.4A, C and E),
251
illustrate the position of M. tuberculosis-GFP within the tissue. From the data presented in Fig.4B it
252
was clear that M. tuberculosis-GFP bacilli were contained within β-III-tubulin-positive neuronal
253
structures (red), indicated by partial co-localisation (yellow) of the red and green fluorescence
254
signals. Similarly, bacilli resided within MAP2 positive neuronal structures (red), which resulted in a
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yellow co-localisation signal (Fig.4D). Green fluorescent bacilli were also present although it was
256
unclear whether the latter represented free or internalised bacilli where MAP2 did not condense
257
around the bacillus; limitation in optical resolution preventing the delineation of cellular structure.
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Microscopic observations indicated that CD11b+ microglial cells contained higher numbers of bacilli
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compared to neurons. CD11b as a cell surface antigen allows for sufficient spatial separation of the
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red signal on the surface and the green signal from the internalised bacilli not to result in a co-
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localised (yellow) signal clearly evident in Fig.4F.
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Therefore the data demonstrates that M. tuberculosis bacilli can be internalised by neurons both
264
under in vitro and in vivo conditions, but that neuronal uptake is subservient to that of microglia.
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M. tuberculosisis bacilli encapsulated within neuronal cytoskeletal structures.
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To gain additional insight into the relationship between neuronal structures and internalised bacilli,
268
localised cytoplasmic sites were further investigatedin vitro and in vivo. Bacilli may display distinct
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condensation of cytoskeletal elements (MAP2+) around it, which resulted in partial co-localisation of
270
the fluorescent signals (yellow), but still allowed for separation of the structures in vitro (Fig.5A).
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Fig.5B confirmed neuronal internalisation of the bacillus, whereas closer inspection and analysis of
272
detailed images (Fig.5B1-B3) revealed that the bacillus resided within a MAP2-positive capsule-like
273
cytoplasmic neuronal structure indicative of rearrangement of the cytoskeleton during infection.
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Similarly, this co-localisation was observed in vivo. As described above, in Fig.4B and Fig.4D, co-
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localisation indicated M. tuberculosisinfection of neurons within the CNS following intra-cerebral
276
challenge. Scan resolution of these sections, due to background and tissue thickness (40µm), was
277
not sufficient to separate the red and green immunofluorescent signals from cytoplasmic markers
278
and bacilli, respectively. However, the MAP2 panel (Fig.5D) showed MAP2 condensation within the
279
area surrounding the bacillus. Therefore, it is plausible that thein vivo infection of neurons by M
280
tuberculosis (Fig.5C-E) likely reflects conditions as observed in vitro (Fig.5B1-B3).
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Cultured human neuronal cells internalise M. tuberculosis bacilli.
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The data demonstrated M. tuberculosis internalisation by murine derived neuronal cell lines and
284
primary murine neuronal cultures, which were confirmed by neuronal uptakein vivo. To ascertain
285
whether these observations have clinical relevance, we infected retinoic acid differentiated SK-N-SH
286
human derived neuroblastoma cell cultures with M. tuberculosis-GFP bacilli at an MOI of 30:1. The
287
cytoplasmic localisation of fluorescent bacilli within SK-N-SH cultures was confirmed through
288
staining with the neuronal specific markers β-III-tubulin or MAP2. Similar to observations described
289
for murine derived neuronal cultures, the orthogonal display of the three dimensional data
290
demonstrated internalised M. tuberculosis bacilli in either β-III-tubulin positive (Fig.6C) or MAP2
291
positive (Fig.6F) cytoplasmic structures. In addition, internalisation was confirmed via the co-
292
localisation of the green (bacilli) and red (β-III-tubulin or MAP2) fluorescence signals resulting in a
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partially yellow fluorescent signal (Fig.6C and 6F). Quantification of the percentage of SK-N-SH cells
294
associated with bacilli showed a statistically significant increase from 10.2% to 21% over 48h (Table
295
1), similar to observations in murine derived neuronal cultures. The percentage of cells, which
296
internalised bacilli increased from 11.9% to 18.5% from 24h to 48h (Table 1). This study therefore
297
confirms that human neuronal cells are capable of being infected by M. tuberculosis bacilli.
298 299
Limited association of M. tuberculosis bacilli with phagolysosomes during neuronal infection
300
Previous studies have demonstrated that neurons are capable of phagocytosis (26) thereby
301
introducing the possibility that M. tuberculosis bacilli may be internalised in this manner. We
302
investigated M. tuberculosis bacilli association with phagolysosomes in primary neuronal cultures
303
infected at a MOI of 30:1 for 24h and 48h, respectively. Analysis of confocal images showed
304
predominant separation of the Lysotracker Red marker and green fluorescent bacilli, which indicated
305
either arrest of phagosome-lysosomal fusion during neuron infection or alternatively, residence of
306
bacilli within the cytosol and not within phagosomes (Fig.7).
307 308
Neurons are activated during M. tuberculosis infection
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Neurons are capable of generating an immune response when confronted by various pathogens.
310
However, neuronal activation in response to M. tuberculosis bacilli has not yet been reported. In
311
this study IL-1β, IL-6 and IL-10 cytokine expression was measured in M. tuberculosis bacilli infected
312
HT22, Neuro-2a and primary neuronsas an indication of functional response (Figure 8). Both
313
neuronal cell lines and primary neuronal cultures had significantly higher (p
