Complement opsonization promotes HSV-2
infection of human dendritic cells
Elisa Crisci, Rada Ellegård, Sofia Nyström, Elin Rondahl, Lena Serrander, Tomas Bergström,
Christopher Sjöwall, Kristina Eriksson and Marie Larsson
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Elisa Crisci, Rada Ellegård, Sofia Nyström, Elin Rondahl, Lena Serrander, Tomas Bergström,
Christopher Sjöwall, Kristina Eriksson and Marie Larsson, Complement opsonization
promotes HSV-2 infection of human dendritic cells, 2016, Journal of Virology, 1-42.
http://dx.doi.org/10.1128/JVI.00224-16
Copyright: American Society for Microbiology
http://www.asm.org/
Postprint available at: Linköping University Electronic Press
Complement opsonization promotes HSV-2 infection of human
1dendritic cells
2Elisa Crisci1, Rada Ellegård1, Sofia Nyström1, Elin Rondahl2, Lena Serrander3, Tomas
3
Bergström4, Christopher Sjöwall5, Kristina Eriksson6 and Marie Larsson1#
4
5
1Division of molecular virology, Department of Clinical and Experimental Medicine, Linköping
6
University, Linköping, Sweden. 2Division of Infectious Diseases, Department of Clinical and
7
Experimental Medicine, Linköping University, Linköping, Sweden, 3Division of Clinical
8
Microbiology, Linköping University Hospital, Linköping, Sweden. 4Department of Infectious
9
Disease, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden. 5AIR
10
Rheumatology, Department of Clinical and Experimental Medicine, Linköping University,
11
Linköping, Sweden. 6Department of Rheumatology and Inflammation Research, University of
12
Gothenburg, Gothenburg, Sweden.
13 14
# Corresponding author: Professor Marie Larsson, marie.larsson@liu.se
15
16
17
Running title: Role of complement in HSV-2 infection of DCs
18
JVI Accepted Manuscript Posted Online 2 March 2016 J. Virol. doi:10.1128/JVI.00224-16
Copyright © 2016 Crisci et al.
Abstract
19
Herpes virus type 2 (HSV2) is one of the most common sexually transmitted infections globally
20
with a very high prevalence in many countries. During HSV2 infection viral particles become
21
coated with complement proteins and antibodies, both existent in the genital fluids, which could
22
influence the activation of the immune responses. In genital mucosa, the primary target cells for
23
HSV2 infection are epithelial cells, but resident immune cells such as dendritic cells (DCs) are
24
also infected. The DCs are the activators of the ensuing immune responses directed against
25
HSV2, and the aim of this study was to examine the effects opsonization of HSV2, either with
26
complement alone or with complement and antibodies, had on the infection of immature DCs
27
and their ability to mount inflammatory and antiviral responses. Complement opsonization of
28
HSV2 enhanced both the direct infection of immature DCs and their production of new
29
infectious viral particles. The enhanced infection required activation of the complement cascade
30
and functional complement receptor 3. Furthermore, HSV2 infection of DCs required
31
endocytosis of viral particles and their delivery into an acid endosomal compartment. The
32
presence of complement in combination with HSV1 or HSV2 specific antibodies more or less
33
abolished the HSV2 infection of DCs.
34
Our results clearly demonstrate the importance of studying HSV2 infection under conditions
35
that ensue in vivo, i.e. when the virions are covered in complement fragments and complement
36
fragments and antibodies, as this will shape the infection and the subsequent immune response
37
and needs to be further elucidated.
38 39
Keywords: HSV2 infection, dendritic cells, complement, antibodies
40
Importance
42 43
During HSV2 infection viral particles should become coated with complement proteins and
44
antibodies, both existent in the genital fluids, which could influence the activation of the immune
45
responses. The dendritic cells are the activators of the immune responses directed against HSV2,
46
and the aim of this study was to examine the effects of complement alone or complement and
47
antibodies, on the HSV2 infection of dendritic cells and their ability to mount inflammatory and
48
antiviral responses.
49
Our results demonstrate that the presence of antibodies and complement in the genital
50
environment can influence HSV2 infection under in vitro conditions that reflect the in vivo
51
situation. We believe that our findings are highly relevant for the understanding of HSV2
52 pathogenesis. 53 54 55 56 57 58 59 60 61 62
Introduction
63
Worldwide, herpes virus type 2 (HSV2) is one of the most common sexually transmitted
64
infections with a high seroprevalence, over 50% in developing countries (1, 2). Many infected
65
individuals are asymptomatic, and shedding of HSV2 in the genital tract can occur without any
66
clinical symptoms (3). Notably, several studies indicate that preexisting genital herpes enhances
67
the acquisition, transmission, and progression of human immunodeficiency virus (HIV-1) (1-4).
68
The innate immune response of the genital tract is the first line of defense against sexually
69
transmitted viruses such as HSV2 (4). In the genital mucosa, epithelial cells are primary targets
70
of HSV2 infection (1), but mucosa immune cells such as dendritic cells (DCs) can also become
71
infected by HSV2 (5). The viral envelope of HSV2 contains an array of viral glycoproteins that
72
are involved in the infection or the immune evasion (6, 7). The HSV2 glycoprotein C (gC) binds
73
complement 3b (C3b) (7-11), which provides protection against complement-mediated virus
74
neutralization, i.e. destruction (9, 12). HSV2 gC facilitates virus entry by attaching the viral
75
particle to host cell-surface heparin sulfate and heparin (13) and the absence of gC sensitizes
76
HSV2 to lysis through the classical complement pathway in epithelial cells (14).
77 78
It’s clear from in vivo studies in different mouse models that the complement pathway plays
79
an important role in the HSV infection (15-17). Complement proteins are present in vaginal
80
secretions (2) and seminal plasma (18, 19) and during a HSV2 infection the viral particles should
81
be complement coated (9, 10), which might influence the infection and activation of the immune
82
responses. Besides complement, the genital secretions contain antibodies which influence the
83
mucosal immune response (20, 21). It is possible that preexisting HSV1 antibodies play a role in
84
protecting individuals from acquiring HSV2 or in the clinical manifestations of the HSV2
infection (22-24). Individuals with HSV1 immunity tend to remain asymptomatic for HSV2 and
86
to have their first clinical manifestation of genital herpes only after experiencing an
87
immunosuppressive event (25).
88
Only few studies exist on the interaction between HSV2 and human DCs and they were
89
performed using monocyte derived DCs (MDDCs) (26, 27). HSV2 induces a productive viral
90
infection in MDDCs (5) and apoptosis in both infected and bystander cells (26). In DCs,
91
infectious HSV2 triggers the release of pro-inflammatory cytokines, most notably TNF-α, but
92
also IL-6 (26, 27) and antiviral factors such as IFN-β (27). Other effects exerted by HSV2 on
93
MDDCs include increased expression of aldehyde dehydrogenase member A1 (27), and impaired
94
antigen presentation (26).
95
The aim of this study was to examine the effects of opsonization of HSV2, i.e. with
96
complement alone or complement and HSV specific antibodies, exerted on the viral infection of
97
immature monocyte derived DCs and these cells’ ability to mount inflammatory and antiviral
98
responses in response to the viral exposure. Complement opsonized HSV2, both by human
99
serum and seminal plasma, gave enhanced infection of DCs and higher productive infection
100
compared to free, non-opsonized, HSV2. Furthermore, opsonization gave rise to significantly
101
higher gene expression of all inflammatory and antiviral factor tested but on the protein level
102
these differences between the free and complement opsonized HSV2 were not as clear as at gene
103
level . The presence of complement and HSV1 or HSV2 specific antibodies decreased infection
104
and inflammation and antiviral responses in the DCs. The HSV2 infection of DCs required
105
endocytosis and endosomal acidification as inhibition of these cellular events decreased the
106
infection. The enhanced infection induced by complement opsonized virions required functional
107
complement receptor 3 (CR3). This work clearly demonstrates the importance to study HSV2
infection under conditions that reflect the in vivo situation, i.e. virions covered in complement
109
fragments or complement fragment and antibodies need to be further explored.
110 111
Materials and methods
112
Reagents
113
Dendritic cell (DC) culture medium RPMI-1640 (GIBCO, Sweden) was supplemented with
114
2mM glutamine, 20µg/ml gentamicin (GIBCO), 10mM HEPES (GIBCO), and 1% human
115
plasma. 100U/ml recombinant human GM-CSF (Genzyme) and 300U/ml recombinant human
116
IL-4 (R&D Systems, Minneapolis, MN, USA) were utilized for in vitro propagation of DCs.
117
Vero cells (ATCC, UK) were cultured in Dulbecco's Modified Eagle's medium (DMEM:
118
GIBCO) with 10% FCS, 2mM glutamine, 20µg/ml gentamicin and 10mM HEPES.
119 120
Propagation of monocyte derived dendritic cells
121
Whole blood was obtained from volunteers as well as from four individuals with a diagnosis of
122
systemic lupus erythematosus (SLE) based on the recent classification criteria (28) with
123
rs1143679 (R77H) mutation in CD11b (29) (Ethical Permits M173-07 and M75-08/2008) as
124
described previously (30). Peripheral blood mononuclear cells (PBMC) were separated by
125
density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway,
126
NJ, USA) and incubated on tissue culture dishes (BD, Europe) for 1h at 37ºC to allow adherence
127
of the DC progenitors before the non-adherent cells were removed by washing with RPMI. The
128
progenitors were differentiated into immature monocyte derived DCs (hereafter referred to as
129
immature DCs) by adding 100U/ml GM-CSF and 300U/ml IL-4 at day 0, 2, and 4 of culture. On
day 5, the DCs were assessed for expression of CD14 and CD83 markers as quality control and
131
then used for experiments.
132
133
Virus propagation and titration
134
HSV2 virus stock was prepared in African green monkey kidney cells (GMK) cultured in Eagles
135
MEM supplemented with 10% FCS as previously described (31). The HSV2 strain used was
136
strain 333. UV inactivation of HSV2 was accomplished by exposing the viruses to UV light for
137 0.5h. 138 139 Opsonization of HSV2 140
Complement opsonization of HSV2 was done by incubation of virions with an equal volume of
141
human serum (HS) or seminal plasma (SP) (Ethical Permits M173-07 and M75-08/2008).
142
Different types of HS were used for virus opsonization: HSV1 and HSV2 seronegative serum
143
opsonized virus (C-HSV2), HSV1 seropositive serum opsonized virus (C1-HSV2) or HSV2
144
seropositive serum opsonized virus (C2-HSV2). The HS was tested for HSV antibodies using
145
HerpeSelect® 1 ELISA IgG and HerpeSelect® 2 ELISA IgG kits (Focus Diagnostics, Cypress,
146
CA, USA). We utilized nine HSV seronegative sera, eight HSV2+ sera, and eight HSV1+ sera
147
for the experiments. For the experiments with seminal plasma, we used samples from four HSV1
148
and HSV2 seronegative donors. Free virus (F-HSV2) was treated with medium alone and mock
149
(medium alone) was used as negative control. Additionally, DCs treated with serum or seminal
150
plasma were used as negative control. All groups were incubated for 1h at 37°C and then directly
151
used in the HSV2 infection experiments. Heat inactivation of the complement was done by
152
incubation of human serum or seminal plasma at 56°C for 1h (HI-C).
154
HSV2 infection of Dendritic cell
155
Immature DCs (0.5 x106) were infected with mock, F-HSV2, C-HSV2, C1-HSV2, or C2-HSV2
156
with multiplicity of infection (MOI) of 1 to 3 for 2h at 37°C in RPMI alone or in 1% plasma
157
from HSV seronegative donors. The different groups of DCs were then washed and cultured in
158
1% plasma from HSV seronegative donors for a total of 6h or 24h. The DCs were harvested,
159
washed, and lysed with Bioline RLY lysis buffer (Bioline, UK) for RNA extraction or fixed with
160
4% paraformaldehyde (PFA) for 10min at 4°C for flow cytometry staining.
161 162
Viral binding and endocytosis
163
To evaluate the binding and uptake of virus particles, DCs were incubated for 2h at 37ºC and at
164
4ºC. Cells were collected, counted, and resuspended in distilled water and stored at -20ºC. To
165
assess the amount of DNA copies viral DNA was extracted with Qiagen, EZ1 Virus Mini 2.0
166
extraction Kit. A specific PCR was performed using Quantifast ROX Vial Kit (Qiagen, Sweden)
167
or Takyon No ROX Probe Master mix dTTP (Eurogentec S.A. Belgium) with HSV2 gG forward
168
primer: AGA TAT CCT CTT TAT CAT CAG CAC CA and HSV2 gG reverse primer: TTG
169
TGC CAA GGC GA and the probe: CAG ACA AAC GAA CGC CG (33).
170
To determine whether the HSV2 infection of DC was dependent on acidification, we used the
171
acidification inhibitors; NH4Cl, (40mM, Sigma), and Bafilomycin A1 (BAF, 50nM; Sigma).
172
Additionally, the requirement of endocytosis was assessed using cytochalasin D (CCD, 10µM;
173
Sigma), clathrin mediated endocytosis was assessed using chlorpromazine (CP, 6.25µg/ml;
174
blocks clathrin mediated entry; Sigma), and protein transport was assessed with monensin (Mon,
175
4µl/ml; BD, Europe). All the agents were used 30min before the infection of the DCs.
177
Assessment of productive HSV2 infection of DCs
178
Supernatants from the HSV2 infection experiments were harvested at 6h and 24h and viral yields
179
were quantified using a modified plaque assay method (35) on Vero cells. Briefly, DC
180
supernatants were incubated in 2 or 10 fold dilution in 24-well plates with a confluent monolayer
181
of Vero cells for 1h at 37°C. After the washing, the plates were coated with complete medium
182
mixed with 2% agarose (1:1) and incubated for additional 3-4 days before assessing plaque
183
forming unites (PFU). Mock and UV-HSV2 were used as negative controls. All samples were
184
tested in duplicates or triplicates.
185 186
Total RNA extraction, reverse transcription and qPCR
187
Total RNA from DCs exposed to mock, F-HSV2, C-HSV2, C1-HSV2, and C2-HSV2 was
188
extracted using Isolate II RNA Mini Kit (Bioline, UK) and total cDNA was produced by
189
SuperScript III Reverse Transcriptase First Strand cDNA Synthesis kit (Invitrogen, Carlsbad,
190
CA, USA). Quantification of gene transcripts was performed using the SensiFAST SYBR®
Hi-191
ROX Kit (Bioline, UK) using a 7900HT Fast Real Time PCR system with 7900 System SDS 2.3
192
Software (Applied Biosystems, Sweden). Primers targeting β-actin and GAPDH were used as
193
housekeeping genes for reference as described by Vandesompele (36). Primers were purchased
194
from CyberGene AB, Stockholm, Sweden (Primer sequences; Supplementary Table 1). To
195
compensate for variation between plates, values were normalized as described by Rieu (37),
196
subtracting each value by the average of all values from the same experiment.
197 198
Assessment of inflammatory factors with ELISA
Levels of TNF-α, IL-6, IFN-α (Mabtech, Sweden), and IFN-β (VeriKine™ Kit, PBL Assay
200
Science, USA) proteins were assessed in supernatants from mock, F-HSV2, C-HSV2, C1-HSV2,
201
and C2-HSV2 24h infected DCs. In addition, these factors were also examined in supernatants
202
from SP opsonized HSV2 infected DCs at 24h and for endocytosis studies at 6h. All the ELISAs
203
were performed following the manufacturer’s instructions.
204 205
Flow cytometry
206
The quality of immature DCs were assessed by staining with anti-human CD83 and CD14 PE
207
conjugated antibodies (BD, Europe). DCs were used if the purity was more than 95% and their
208
expression of CD14 and CD83 were less than 10%. To evaluate the level of HSV2 infection in
209
immature DCs, cells were permeabilized with PBS containing 0.2% saponin and 0.2% FCS and
210
incubated with polyclonal Ab (pAb) against HSV2 (identifying all major glycoproteins in the
211
viral envelope and at least one core protein; B0116, DAKO, Denmark) or monoclonal Ab (mAb)
212
against HSV2 ICP8 (4E6) (Santa Cruz biotechnology, USA) followed by FITC or Alexa 488
213
conjugated secondary Ab (Abcam, Europe). Zombie Aqua™ Fixable Viability Kit (BioLegend,
214
Europe) was used to discern viable/dead cells. Additionally, PE conjugated mAb against
215
CD11b/Mac-1 (BD, Europe) was used to evaluate the presence of CD11b receptor on DCs. The
216
samples were assessed by flow cytometry (FACS Canto, BD) and analyzed by FlowJo (Treestar,
217
Ashland, OR, USA).
218
219
CD11b protein knockdown in dendritic cells by siRNA
220
Knockdown of the CD11b protein with siRNA in immature DCs was done by transfecting
221
immature DCs at day 2 or 3 of culture with siRNA using the transfection reagent DF4
(Dharmacon) or HiPerFect (Qiagen) respectively. The transfection reagents were removed and
223
the cells were used for experiments 2 days after transfection. The siRNA (SMART pool;
224
Dharmacon) was specific for CD11b (Human ITGAM M-008008-01). Non targeting siRNA
225
control pool (D-001206-13-05; Dharmacon) served as control. The transfection efficiency was
226
determined by flow cytometry of cells transfected with siGLO RISC-Free Control siRNA
(D-227
001600-01; Dharmacon). Silencing of CD11b expression was verified by real-time PCR and
228 flow cytometry. 229 230 Statistical analysis 231
The statistical program GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) was used for
232
analysis of all data. Repeated measures ANOVA followed by a Bonferroni post-test or a
two-233
tailed paired t-test were used to test for statistical significance. Results were considered
234
statistically significant if p<0.05. All experiments were performed a minimum of four times
235
using cells derived from different blood donors or different cell line passages. When
236
experimental values were normalized, the mean of free virus or mock were set to 1. qPCR results
237
were normalized for variation between plates as previously described (37). In brief, each value
238
was subtracted by the average of all values and then the obtained values were divided by the
239
average of mock or free virus depending on the different experiments.
240 241
Results
242
Complement opsonization of HSV2 increased infection of immature dendritic cells whereas
243
the presence of neutralizing HSV1 or HSV2 abs abolished the infection
HSV2 has the ability to infect immature monocyte derived DCs (26, 27) and give rise to a low
245
level of productive infection (26). HSV2 expresses gC on the viral envelope and this is a
246
glycoprotein which neutralizes complement factor C3 (7-11). Here we have assessed how
247
complement opsonization affected the HSV2 infection of immature monocyte derived DCs
248
(hereafter referred to as immature DCs) by infecting them with mock, free HSV2 (F-HSV2),
249
HSV2 opsonized with complement using serum negative for HSV1 and HSV2 antibodies
(C-250
HSV2), serum from HSV1 seropositive (C1-HSV2), or HSV2 seropositive (C2-HSV2). The
251
mRNA expression levels of HSV2 thymidine kinase (TK), an enzyme involved in viral
252
nucleotide biosynthesis and DNA metabolism, and glycoprotein D (gD), a receptor part of the
253
core fusion machinery and important in viral entry, were assessed by qPCR at 6h and 24h post
254
infection (Figure 1A-B). C-HSV2 induced significantly higher mRNA expression of HSV2 TK
255
and gD at both 6h and 24h in DCs compared to free virus (Figure 1A-B). The presence of HSV1
256
or HSV2 specific antibodies in the serum used for opsonization almost abolished the infection as
257
seen by the decreased mRNA expression levels of TK and gD compared to the free virus and
C-258
HSV2 at both time points (Figure 1A-B). The gene expression pattern of HSV2 protein ICP0
259
was similar to TK and gD (Supplementary figure 1A). The mRNA profiles matched the
260
profiles of HSV2 proteins expressed at 24h as assessed by flow cytometry, with 2-fold higher
261
levels of HSV2+ cells in the C-HSV2 infected group compared to free HSV2 (Figure 1C-D). A
262
similar protein expression pattern was obtained for HSV2 infected cell protein 8 (ICP8)
263
(Supplementary figure 1B). To verify the results with a source of complement that exists at the
264
site of infection, we assessed the effect opsonization with seminal plasma (SP) exerted on the
265
HSV2 infection of DCs. The SP opsonized HSV2 (SP-HSV2) induced a higher infection of DCs
266
compared to free virus at both 6h and 24h (Figure 1E) at gene transcripts level. This indicated
that even if less complement was present in the seminal plasma (32) the complement opsonized
268
virus still had higher capacity to infect DC than free virus.
269
HSV2 infection is known to induce apoptosis in DCs (5) and we could conform this effects in
270
our system with higher levels of dead and apoptotic cells for F-HSV2 and C-HSV2 compared to
271
C1-HSV2, C2-HSV2, and mock cells (Supplementary figure 1C and data not shown). As an
272
infection control, we assessed the effects of UV inactivated HSV2 (UV) and found that this
273
inactivation abolished the infection (Supplementary figure 2), which is consistent with previous
274
findings (26).
275 276
Complement opsonization of HSV2 enhanced the productive infection of dendritic cells
277
To assess the levels of productive HSV2 infection in DCs we measured the amount of released
278
infectious virions in the supernatants at 24h post infection by plaque forming assay. After 24h,
279
immature DCs infected with C-HSV2 had significantly higher production and release of viral
280
particles compared to free virus, C1-HSV2, and C2-HSV2 (Figure 1F). A similar pattern was
281
obtained when HSV2 was opsonized with seminal plasma (Figure 1G).
282
283
Complement opsonization of HSV2 increased inflammatory and antiviral transcripts in
284
dendritic cells.
285
Seeing that complement opsonized HSV2 enhanced the infection we assessed the effects the
286
opsonized virions had on the ability of DCs to take the action against the infection by producing
287
antiviral and inflammatory factors. We have recently demonstrated enhanced infection and
288
suppressed antiviral and inflammatory responses in DCs exposed to complement opsonized
HIV-289
1 (30). The mRNA expression levels of antiviral factors IFN-β, IFN-α, and MX1 and
inflammatory factors TNF-α, IL-6, and IL-1β were assessed for free and opsonized HSV2. The
291
mRNA levels of IFN-β and MX1 induced by C-HSV2 were significantly higher compared
F-292
HSV2 at 24h (Figure 2A), but no significant difference was observed for IFN-α levels between
293
F-HSV2 and C-HSV2 (Figure 2A). The presence of HSV1 or HSV2 specific antibodies
294
decreased IFN-β expression at 24h (Figure 2A). Surprisingly, there were similar protein levels
295
of the antiviral factors, IFN-β and IFN-α, produced by DCs after F-HSV2 and C-HSV2 infection
296
(Figure 2B), whereas the protein levels were significantly lower for C1-HSV2 and C2-HSV2
297
compared to F-HSV2 (Figure 2B). C-HSV2 significantly increased the mRNA levels of the
298
inflammatory factors TNF-α, IL-6 and IL1-β in DCs at 24h compared to F-HSV2 (Figure 2C).
299
In addition, the mRNA expression of the chemokines CCL3 and CXCL8 had similar pattern as
300
the inflammatory factors at 24h post infection (Supplementary Figure 3). The secretion of IL-6
301
and TNF-α in DC supernatants were assessed at 24h post infection. Complement opsonized virus
302
and F-HSV2 induced similar levels of these inflammatory factors (Figure 2D), The reason for
303
this discrepancy between mRNA and protein expression levels could be due to a more
304
suppressive cellular function in C-HSV2 infected DCs compared to free virus and/or activation
305
of a different regulation of protein transcription by microRNAs in C-HSV2 compared to free
306
HSV2 infected DCs. The TNF-α levels induced by C-HSV2 were much higher than the levels for
307
C1-HSV2 and C2-HSV2 (Figure 2D). Moreover, similar to serum, HSV2 opsonized with
308
seminal plasma induced higher TNF-α and IFN-β mRNA levels in DCs compared to free HSV2
309
(Figure 2E). At protein level seminal plasma opsonized virus tend to increase the secretion of
310
TNF-α and IFN-β compared to free virus, but the data were not statistically significant, and
311
exhibited a big variation between donors (Figure 2F).
312 313
Complement opsonization did not alter the amount of HSV2 particles binding to and taken
314
up by dendritic cells
315
To examine whether the higher level of infection induced by C-HSV2 was due to altered binding
316
and internalization mechanisms, the levels of bound and internalized F-HSV2, C-HSV2,
C1-317
HSV2, C2-HSV2 and heat inactivated C-HSV2 (HI-C) in DCs were assessed measuring the viral
318
DNA copies/ml after 2h at 4°C (data not shown) or 37°C (Figure 3A). The levels of bound and
319
internalized virions were similar for all virus groups with a tendency of higher viral uptake for
320
C1-HSV2 and C2-HSV2 (Figure 3A).
321 322
Inhibition of endocytosis and endosomal acidification impeded complement opsonized
323
HSV2 infection of dendritic cells
324
HSV infection of epithelial cell lines has previously been shown to in part require an acid
325
endosomal compartment and endocytosis (39, 40), therefore we examined the effects that
326
inhibitors of acidification NH4Cl, and Bafilomycin A1 (BAF), inhibitors of endocytosis
327
Cytochalasin D (CCD) and chlorpromazine (CP), and monensin (Mon) a carboxylic ionophore
328
exerted on the HSV2 infection of DCs. Drugs were used at concentrations previously shown to
329
block viral uptake and endosomal acidification in DCs or other cell types (34, 41-43) without
330
affecting the viral infectivity. Neutralization of endosomal acidification with NH4Cl decreased
331
the F-HSV2 and C-HSV2 infection of DCs, with almost 20-fold decreased C-HSV2 infection at
332
6h post infection (Figure 3B). Moreover, mRNA expression level of the antiviral response, as
333
measured by IFN-β, gave the same profile as the infection with significantly decreased C-HSV2
334
levels (Figure 3B). The inhibition of DC endocytosis with CCD more or less abolished the
HSV2 and C-HSV2 infection at 6 hours (Figure 3C) and the same effects were seen for the
336
antiviral response (Figure 3C).
337
F-HSV2 infectivity and antiviral response were also inhibited by monensin, chlorpromazine, and
338
bafilomycin (Figure 3D), further confirming the involvement of endosomal acidification and
339
clathrin mediated endocytosis in the HSV2 infection process of DCs. Our finding regarding the
340
effect of bafilomycin on HSV2 infection correlated with a previous finding (43). C-HSV2’s
341
infectivity was also inhibited by monensin and chlorpromazine but only to a significant level by
342
monensin, whereas bafilomycin had no effect (Figure 3E). The inhibitors decreased also the
343
antiviral response induced by C-HSV2 (Figure 3E). The treatment with these inhibitors had no
344
direct effect on the viral infectivity, i.e. the ability of HSV2 to infect, or on the DCs’ baseline
345
expression of antiviral and inflammatory factors (data not shown).
346 347
The elevated HSV2 infection in dendritic cells induced by complement opsonized virions
348
required functional complement activation
349
The activation of the complement cascade is inhibited by heat inactivation of the serum and we
350
confirmed the involvement of complement in the enhanced infection seen for C-HSV2 by using
351
heat inactivated serum. Opsonization of HSV2 with heat inactivated serum gave the same
352
binding and internalization level as F-HSV2 and C-HSV2 (data not shown). Heat inactivation of
353
complement opsonized virus significantly decreased the mRNA expression of HSV2 TK and
354
HSV2 gD compared to C-HSV2 and the levels were similar to free virus at 6h (Figure 4A). The
355
same pattern was seen for the productive infection (Figure 4A) and for HSV2 protein expression
356
at 24h (Figure 4B). The inactivation had no significant effects on C1-HSV2 and C2-HSV2
357
infection levels, nor on TK and gD mRNA or HSV2 protein levels (Figure 4B). In the case of
antiviral and inflammatory factors, the pattern was similar and HSV2 opsonized with heat
359
inactivated serum restored IFN-β and TNF-α to the levels of free virus at 24h (Figure 4C).
360
361
The elevated infection induced by complement opsonized HSV2 in dendritic cells required
362
functional complement receptor 3
363
Complement receptor 3 (CR3) is exploited by several pathogens for suppression of innate
364
responses (44, 45). DC expression of CR3 is required for complement opsonized HIV’s
365
augmentation of infection (30), consequently we examined if the enhanced HSV2 infection of
366
DCs by complement opsonized HSV2 also was dependent on viral binding to CR3. Here we used
367
DCs derived from individuals with SLE with mutated CR3 alpha-integrin CD11b (rs1143697:
368
R77H), which gives decreased expression of CD11b and impairs the signaling through CR3 (30,
369
46, 47). The free HSV2 gave the same level of infection in DCs derived from individuals with
370
SLE as in cell from healthy donors (Supplementary figure 4). The enhanced infection seen for
371
C-HSV2 in DCs from healthy individuals was abolished when the DCs had dysfunctional CR3
372
(Figure 5A). Moreover, the enhanced gene expression of inflammatory and antiviral factors
373
normally seen for C-HSV2 exposed DCs was dot detected when DCs with mutated CR3 were
374
used (Figure 5B). To confirm the findings from the SLE patient derived DCs with dysfunctional
375
CR3, we knocked down CD11b expression with siRNA and assessed infection, inflammation
376
and antiviral responses (Figure 5D). The knockdown of CD11b abolished the increased
C-377
HSV2 infection, whereas this increase was still present in the control siRNA transfected cells
378
(Figure 5C). Moreover, the higher gene expression of inflammatory and antiviral factors seen
379
for C-HSV2 exposed DCs was not present in the CD11b knockdown DCs (Figure 5C).
380 381
Discussion
382
Langerhans cells (LCs) and DCs are one of the initial responder cells during the establishment
383
of HSV2 infection in the mucosa and are involved in the induction of the HSV2 specific adaptive
384
immunity via DC cross presentation (5, 48). At the site of infection, the HSV2 virions are
385
exposed to soluble factors, such as complement and antibodies, which should influence the
386
effects virions exert on their target cells as well as the local infection. We found that HSV2’s
387
capacity to utilize the complement system, using serum or seminal plasma, enhanced the virus’
388
ability to directly infect DCs and that the enhanced infection required functional CR3. Presence
389
of HSV1 or HSV2 specific antibodies during the opsonization more or less abolished the ability
390
of HSV2 to infect the DCs. The HSV2 infection of DCs required endocytosis of virus particles
391
and acidification of the endosomal compartment. Complement opsonized HSV2 induced higher
392
mRNA expression but not statistically significant higher protein secretion of antiviral and
393
inflammatory factors in the DCs compared to free HSV2.
394
HSV2 has the ability to infect several cell types such as epithelial cells, nerve cells and
395
DCs located in the genital mucosa (1, 5) and in vitro studies have shown that immature
396
monocyte derived DCs and LCs are highly susceptible to HSV2 infection (49-51). Our findings
397
support that HSV2 infects DCs and that this gives rise to a low level of productive infection. The
398
low level of productive infection is not surprising since previous studies have found HSV2 and
399
HSV1 infection of DCs to be predominantly abortive (26, 52-54). This should be the explanation
400
for the discrepancies we see with between the high levels of viral mRNA transcripts and the low
401
levels of infectious HSV2 produced by infected DCs. One additional explanation could be the
402
existence of cellular inhibitors of infection that influence events after the mRNA transcription,
403
i.e. inhibiting the production of viral proteins and/or infective virions.
The first steps in the in the HSV infection of target cells, i.e. binding and uptake of viral
405
particles, have been investigated and almost all studies focused on HSV1 and epithelial cell lines
406
(34, 39, 40, 55). The mechanism for HSV1 and HSV2 uptake in epithelial cells is rapid
407
endocytosis (34). The HSV1 endocytosis requires several of the viral envelope glycoproteins, i.e.
408
gB, gD, and gH-gL (56, 57) and in keratinocytes HSV1 uptake is a cholesterol and dynamin
409
mediated process. In the endosomal compartment HSV1 and HSV2 require the gD receptor to
410
access the host cell cytosol (34) and in the case for HSV1 dynamin to be able to infect (58). The
411
binding and uptake of HSV2 by DCs has not been examined previously, and we found that
412
complement opsonization had no effect on the amount of HSV2 virions bound or internalized.
413
The HSV2 infection for both free and complement opsonized virions was dependent on
414
endocytosis and endosomal acidification. Interestingly, the infection with free virus involved
415
clathrin dependent endocytosis, whereas complement opsonized virus seemed to utilize a clathrin
416
independent mechanism for infection. In addition to suppressing infection, the neutralization of
417
endosomal acidification decreased the antiviral responses in DCs. This indicates that the antiviral
418
responses require a degradation of the viral particles and/or active infection of the DCs. TLR3
419
and TLR2 have been suggested to be as important PRRs in the immunological control of the
420
HSV2 infection (59-62) Furthermore, sensors such as STING and DAI can also sense HSV2
421
(63). The exact PRRs involved in the activation of the antiviral and inflammatory responses in
422
the human DCs in our system of HSV2 infection, remain to be determined. Our finding that
423
HSV2 antiviral responses in DCs required endocytosis of the virus into an acidified endosomal
424
compartment, suggests the involvement of endosomal and/or cytosolic PRRs.
425
A number of earlier studies have suggested that a prior oral HSV1 infection can provide
426
partial protection against genital HSV2 acquisition (22-24), whereas others have proposed that it
has no protective effect (64-66). At present, most evidence indicates that preexisting HSV1
428
antibodies do not inhibit the infection, rather they will make the HSV2 infection less pathogenic
429
with milder symptoms (22-25). We found that the presence of HSV1 specific antibodies more or
430
less abolished the ability of HSV2 to infect DCs and surprisingly they neutralized the infection
431
with the same efficiency as HSV2 specific antibodies. The presence of HSV1 or HSV2 432
antibodies did not decrease the uptake of virus by DCs, rather slightly increased it, so the
433
absence of infection was not due to inability of the virus to bind and be internalized but rather to
434
neutralization of HSV2 infectivity. This raises questions such as how the adaptive immune
435
response induced by a prior oral HSV1 infection, affects the genital mucosal responses during
436
primary genital HSV2 infection, i.e. whether the existing HSV1 antibodies will neutralize the
437
virus and render the virions less cytopathic and suppressive for the DCs functionality and this
438
needs further elucidation.
439
HSV2 uses many strategies to establish persistent infection and inhibition of complement
440
activation/cascade and antibody binding by gC and gE/gI proteins on the viral surface are part of
441
these mechanisms (7, 67). gC on HSV2 (gC2) and HSV1 (gC1) both bind C3b (8-10) and HSV1
442
gC also interferes with the binding of C5 and properdin to C3b (12, 15, 68, 69 ). In fact, HSV1
443
gC1 contains a C5- and P-interacting domain that accelerates the decay of the alternative
444
complement pathway C3 convertase and this domain is important in modulating complement
445
activity, since HSV1 lacking this domain is more readily neutralized by complement alone, and
446
is significantly less virulent than wild-type virus in vivo. Interestingly, this domain is absent in
447
HSV2 gC2, suggesting that the mechanism by which HSV2 evades the complement cascade may
448
be distinct from that of HSV1 (12, 14, 70).
In our study we found that complement fragments binding to the HSV2 enhanced the direct
450
and productive infection of DCs. The enhanced HSV2 infection of DCs induced when the virus
451
is complement opsonized has also been observed for HIV-1 by our group and by others (30, 71,
452
72). In the case for HIV-1 and the bacteria Francisella tularensis, the complement opsonization
453
suppresses the pathogen induced antiviral and inflammatory immune response by CR3 mediated
454
modulation of TLR signaling pathways (30, 44). Even if the enhanced infection achieved by
455
complement opsonized HSV2 was due to CR3 interaction, it did not suppress the secretion of
456
antiviral and inflammatory factors. The mechanisms behind these differences between different
457
pathogens could be diversity in receptors, such as type of pattern recognition receptors activated,
458
due to presence of different PAMPS, in combination with the CR3, giving rise to distinct
459
signaling and activation of the DCs.
460
The HSV2 infection of DCs in our hands induced an array of inflammatory and antiviral factors
461
with higher mRNA levels induced by the complement opsonized viruses compared to free virus,
462
whereas the protein expression levels were the same. The reason for these discrepancies between
463
mRNA and protein expression levels could be due to a more suppressive cellular function in
C-464
HSV2 infected DCs compared to free virus and/or activation of a different regulation of protein
465
transcription by microRNAs in C-HSV2 versus free HSV2 infected DCs.
466
In conclusion, the HSV2 exploitation of our innate defense, i.e. the complement system,
467
enhanced the virus ability to infect DCs. Presence of HSV antibodies clearly render the virus
468
virtually noninfectious and less toxic to the DCs and should function as a source of antigens for
469
activation of adaptive immune responses. We clearly demonstrate the importance to study HSV2
470
infection under conditions that reflect the in vivo situation, i.e. virions covered in complement
fragments or complement fragments and antibodies, as these factors will have a profound effect
472
on the virus’ interaction with the host.
Acknowledgements
474
This work has been supported by: AI52731, The Swedish Research Council, The Swedish
475
Physicians against AIDS Research Foundation, The Swedish International Development
476
Cooperation Agency, SIDA SARC, VINNMER for Vinnova, Linköping University Hospital
477
Research Fund, CALF, and The Swedish Society of Medicine for ML. The Swedish Society for
478
Medical Research for CS.
479
480
481
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687 688
Figure Legends
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Figure 1. Complement opsonization of HSV2 increased infection in immature DCs.
690
Immature DCs (106/ml) were exposed to mock or 3 MOI of free HSV2 (F), HSV2 complement
691
opsonized with HSV1/2 seronegative serum (C), HSV2 opsonized with HSV1 (C1), HSV2 (C2)
692
seropositive serum or HSV2 opsonized with seminal plasma (SP) from HSV1/2 seronegative
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donor for 6h or 24h. (A-B) mRNA expression levels at 6h and 24h for HSV2 TK and gD were
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assessed by qPCR. qPCR values were normalized with free virus mean values set to 1. (C-D)
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HSV2 infection was assessed by flow cytometry at 24h using a pAb against HSV2, and
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representative dot plots with percentage of HSV2 positive cells are shown (mean + SEM). Data
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are shown as box and whiskersof 4-6 independent experiments. (E) PFU assays were performed
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to assess productive HSV2 infection, i.e. production of infectious viral particles by DCs.
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Supernatants (24h) from DCs exposed to mock, F, C, C1 and C2 were tested and HSV2 PFU/ml
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calculated. A representative experiment (left) and normalized experiments (N=5) with PFU
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values of free virus values set to 1 (right). Data are shown as box and whisker plots of 5
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independent experiments. (F) mRNA expression levels at 6h and 24h for HSV2 TK and gD were
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assessed by qPCR. qPCR values were normalized with free virus mean values set to 1. (G) PFU
704
assays were performed to assess productive HSV2 infection in DCs. Supernatants (24h) from
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DCs exposed to F and SP were tested and HSV2 PFU/ml calculated. Normalized experiments
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(N=4) with PFU values of free virus values set to 1. *p<0.05. **p<0.005. ***p<0.0005.
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Figure 2. Complement opsonized HSV2 induced higher mRNA levels of inflammatory and
709
antiviral factors in immature DCs compared to free virus.
DCs (106/ml) were exposed to 3 MOI of free HSV2 (F), complement opsonized HSV2 (C),
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HSV2 opsonized with both complement and specific antibodies against HSV1 (C1), or HSV2
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(C2) or mock treated for 24h. mRNA expression of antiviral factors IFN-β, IFN-α, and MX1 (A)
713
and inflammatory factors TNF-α, IL-6, and IL-1β (C) was determined by qPCR. qPCR values
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were normalized with mock mean values set to 1. Protein levels of IFN-α and IFN-β (B) and
IL-715
6 and TNF-α (D) were assessed in the supernatants from DCs by ELISA. Data are shown as box
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and whisker plotsof 5-6 independent experiments. (E) DCs (106/ml) were exposed to 3 MOI of
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free HSV2 (F), complement opsonized HSV2 (C), HSV2 opsonized with seminal plasma (SP)
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from HSV1/2 seronegative donor or mock treated for 24h. mRNA expression of inflammatory
719
factor TNF-α and antiviral factor IFN-β were determined by qPCR. qPCR values were
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normalized with mock mean values set to 1. Data are shown as box and whisker plotsof 5-6
721
independent experiments.* p<0.05. **p<0.005. ***p<0.0005.
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723
Figure 3. HSV2 infection of DCs required endocytosis and endosomal acidification
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DCs (106/ml) were exposed to 3 MOI of free HSV2 (F), complement opsonized HSV2 (C),
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HSV2 opsonized with both complement and specific antibodies against HSV1 (C1) or HSV2
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(C2), heat inactivated C-HSV2 (HI-C) or mock treated for 2h or 6h. (A) Levels of HSV2 viral
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DNA copies in immature DCs exposed to F, C, C1, C2 or HI-C for 2h were assessed by qPCR.
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(B-E) DCs were pre incubated for 30 min with 40mM Ammonium Chloride (NH4Cl) (B), 10µM
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Cytochalasin D (CCD) (C), 50nM Bafilomycin A1 (BAF), 4µl/ml Monensin (Mon) and
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6.25µg/ml Chlorpromazine (CP) (D-E) before HSV2 infection. (B-C) mRNA expression levels
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of HSV2 TK and IFN-β were determined by qPCR. Values have been normalized with free virus
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mean values set to 1 and data are shown as mean+SEM of 4-5 independent experiments. (D-E)
