Abstract
Anaplasma phagocytophilum, the causative agent of human granulocytic
anaplasmosis (HGA), is an obligate intracellular bacterium transmitted
by the bite of black-legged ticks, Ixodes scapularis. The main host
cells in vertebrates are neutrophils. However, the first site of entry
is in the skin during tick feeding. Given that the initial responses
within skin are a crucial determinant of disease outcome in
vector-borne diseases, we used a non-biased approach to characterize
the transcriptional changes that take place at the bite during I.
scapularis feeding and A. phagocytophilum transmission. Experimentally
infected ticks were allowed to feed for 3 days on C57BL/6J mice to
allow bacterial transmission and establishment. Skin biopsies were
taken from the attachment site of uninfected ticks and A.
phagocytophilum-infected ticks. Skin without ticks (intact skin) was
used as baseline. RNA was isolated and sequenced using next-generation
sequencing (NGS). The differentially expressed genes were used to
identify over-represented pathways by gene ontology (GO) and pathway
enrichment (PE). Anaplasma phagocytophilum transmission resulted in the
activation of interferon signaling and neutrophil chemotaxis pathways
in the skin. Interestingly, it also led to the downregulation of genes
encoding extracellular matrix (ECM) components, and upregulation of
metalloproteinases, suggesting that A. phagocytophilum delays wound
healing responses and may increase vascular permeability at the bite
site.
Keywords: transmission, ticks, innate immune responses,
metalloproteinases, extracellular matrix, neutrophils, cytokines,
intracellular bacteria
1. Introduction
The black-legged tick, Ixodes scapularis, has been spreading in recent
years to more locations across the northeastern and midwestern United
States. It is a competent vector of seven different pathogens, known to
cause illness in humans, including Anaplasma phagocytophilum [[36]1].
Anaplasma phagocytophilum is the causative agent of human granulocytic
anaplasmosis (HGA), formerly known as human granulocytic ehrlichiosis
(HGE), which was discovered infecting a cluster of men in the upper
Midwest in 1994 [[37]2]. Later, similar cases were reported in other
parts of the U.S. where Lyme disease is prevalent [[38]3]. Most
recently, 5655 cases were reported to the CDC in 2019, making HGA the
second most common tick-borne disease in the U.S. [[39]4]. In most
cases, the illness is self-limiting, and patients will recover with or
without antibiotic treatment. Symptoms will typically develop 1–2 weeks
following a bite by an infected tick, can lead to hospitalization in
around 36% of confirmed cases, and presents mortality rates of around
0.2–1% [[40]5].
Anaplasma phagocytophilum is a Gram-negative bacterium of small size
(0.4 to 1.5 μm) that replicates inside neutrophils within small
vacuoles termed “morulae” [[41]6]. This bacterium lacks several of the
enzymes involved in peptidoglycan and lipopolysaccharide biosynthesis
[[42]6,[43]7], which are two pathogen-associated molecular patterns
(PAMPs) commonly recognized by vertebrate immune responses.
Nevertheless, stimulation of peripheral blood leukocytes from healthy
donors with cell free bacteria or recombinant A. phagocytophilum outer
protein P44 (rP44) induces the expression of proinflammatory cytokines
[[44]8]. Similarly, high levels of proinflammatory cytokines, such as
interferon (Ifn)-γ, interleukin (IL)-12p70, and IL-10, are detected in
HGA patients [[45]9]. Further, the elevated levels of these cytokines
appear to be associated with the severity and pathology of the disease
in humans and murine models [[46]9,[47]10,[48]11]. Ifn-γ is involved in
the control of A. phagocytophilum infection in mice
[[49]10,[50]11,[51]12] and during the in vitro infection of Hoxb8
neutrophils [[52]13]. In vivo experiments using murine models indicate
that secretion of Inf-γ correlates with Stat1 phosphorylation [[53]14].
Stat1 knock-out in mice leads to increased bacterial loads, severe
disease pathology, increased spleen size, higher cytokines/chemokines
levels in plasma, and reduced iNOS induction [[54]15]. These studies
were focused on systemic immune responses, and little is known about
the immune events that take place during A. phagocytophilum
transmission at the bite site.
Despite the importance of the skin as the site of entry and
establishment of A. phagocytophilum, only a few experiments have been
conducted to understand the events that occur at the bite site. In
2010, Granquist et al. [[55]16] described the site of A.
phagocytophilum transmission in lambs naturally infested with Ixodes
ricinus ticks. Histological inspection of the bite sites showed
evidence of inflammation, accumulation of immune cells such as
neutrophils, macrophages, and other mononuclear cells, and the
deterioration of the collagen matrix. Bacteria were associated with
neutrophils and macrophages. However, the time of feeding for each tick
could not be determined since animals were naturally infested. A later
study similarly reported the presence of A. phagocytophilum within
neutrophils in the skin of experimentally infected sheep later infested
with ticks [[56]17]. This study also reported significantly higher
numbers of neutrophils in the skin of the experimentally infected sheep
independent of the presence of feeding ticks, suggesting that A.
phagocytophilum infection increases migration of neutrophils to the
skin. Although this study considered the changes in expression of eight
immune-related genes, the responses by each animal were variable and no
conclusions on the activation of immune signaling pathways could be
drawn. Thus, an important knowledge gap exists on the signaling and
immunological events that take place during transmission of A.
phagocytophilum by ticks.
The present study explores the gene expression changes that occur at
the bite site during the transmission of A. phagocytophilum. We
describe an upregulation in genes involved in Ifn-γ signaling, defense
responses to viruses, neutrophil chemotaxis, and interleukin-1
responses. Interestingly, A. phagocytophilum transmission appears to
decrease the expression of genes implicated in extracellular matrix
(ECM) organization and wound healing responses. Given that the skin is
the initial site of A. phagocytophilum establishment, an understanding
of the immunological events that take place during initial infection
will help us uncover potential signaling pathways and immune responses
that can be exploited to stop infection.
2. Materials and Methods
2.1. Anaplasma Phagocytophilum Culture
HL60 cells (CCL-240™) were obtained from ATCC (Manassas, VA, USA). HL60
cell cultures were maintained in RPMI media (Corning, Manassas, VA,
USA) supplemented with 10% fetal bovine serum (Gibco, Whaltham, MA,
USA), 1% Glutamax (Gibco, Whaltham, MA, USA), and 1% amphotericin B
(Corning, Manassas, VA, USA) and incubated at 37 °C with 5% CO[2], as
previously described [[57]18]. Cells were maintained until the cell
density became optimal for passage or for infection with A.
phagocytophilum (~1 to 5 × 10^5 cells/mL). Cells were passaged as
follows: 2 mL of cell culture was transferred into a 25 cm^2 tissue
culture flask (Fisher Scientific, Pittsburgh, PA, USA) and supplemented
with 18 mL of freshly prepared culture media. This was repeated every 3
to 5 days, until cells reached passage 10 when they were discarded, and
a new culture was recovered from liquid nitrogen (LN[2]).
For infection with A. phagocytophilum, 2 mL of uninfected HL60 cell
cultures (at approximately 5 × 10^5 cells/mL) was inoculated with 500
μL of A. phagocytophilum-infected HL60 cells (at approximately 2 × 10^5
cells/mL, with ~90% infection). Anaplasma phagocytophilum was cultured
in HL60 cells for up to 5 days. Infections were monitored by placing 1
mL of the suspended cell culture onto a microscope slide, and spinning
with a CytoSpin 4 (Thermo Scientific, Whaltham, MA, USA) at 800× g for
5 min. The infected cells were stained using the Richard-Allan
Scientific™ Three-Step Stain Kit (Thermo Scientific, Whaltham, MA,
USA), according to manufacturer’s specifications. The morulae within
cells were observed by light microscopy with an Olympus model BX43F
(Shinjuku City, Tokyo, Japan). Infections were passaged when the
percentage of infection was greater than 90%, determined by counting
100 HL60 cells with observable morulae. Bacterial cultures were
maintained for up to 5 passages before freezing or infecting mice,
using the procedures described below.
2.2. Mice Infections
C3H/HeJ male mice of 6 weeks of age (The Jackson Laboratory, Bar
Harbor, ME, USA) were used for pathogen acquisition due to their high
susceptibility to infection from Gram-negative bacteria, including to
A. phagocytophilum infection [[58]19]. The susceptibility of this mouse
strain to Gram-negative bacteria is associated with a mutation in the
cytoplasmic domain of Toll-like receptor 4 (TLR4) [[59]20] and has
shown impaired inflammatory and innate immune responses under several
conditions [[60]21,[61]22]. Mice were injected intraperitoneally (i.p.)
with 100 μL A. phagocytophilum-infected HL60 containing 1 × 10^7
bacteria, using 27-gauge needles ([62]Figure 1a). Cells were spun down
at 300× g for 10 min, culture media was removed, and the cells were
suspended in 1× PBS. The number of bacteria was estimated using the
previously described formula [[63]23]. Control mice received an
injection of 100 μL 1× PBS.
Figure 1.
[64]Figure 1
[65]Open in a new tab
Schematic representation of acquisition and transmission feedings. (a)
C3H/HeJ mice were inoculated with Anaplasma phagocytophilum-infected
HL60 cells or PBS (control mice). Blood samples were taken on days 3,
5, and 7, and infection (or lack of) was confirmed by PCR. Animals were
infested with pathogen-free certified larvae on day 8 once infectious
status had been confirmed. Engorged larvae were recovered starting at
day 10 (3 days after infestation in mice) until day 13 (5 days post
infestation in mice). Larvae were allowed to molt for 1 month and
nymphs were tested for A. phagocytophilum infection by PCR and gel
electrophoresis. (b) After confirmation of infection (or lack of),
nymphs were infested into C57BL/6J and were allowed to feed for 3 days.
Skin biopsies were collected from the bite site of Anaplasma
phagocytophilum-infected ticks, uninfected ticks, and from intact skin.
Illustration was created using BioRender.
To confirm infections, cheek bleeds were performed on the mice on days
3, 5, and 7 post-infection (p.i.), collecting 20 to 100 μL of blood in
microvettes 500 K3E (Sarstedt, Nümbercht, Germany) after anesthesia
with 1.25% to 2% isoflurane ([66]Figure 1a). Blood was used for DNA
extraction with the DNeasy Blood & Tissue Kit (QIAGEN, Hilden,
Germany), following the manufacturer’s instructions. DNA quantity and
quality was assessed using a NanoQuant Infinite M200 Pro (Tecan,
Switzerland). PCR amplification of mouse actin was performed to confirm
the absence of contaminants and PCR inhibitors. PCR analyses of the A.
phagocytophilum rpoB and the 16s rRNA genes were performed on the blood
to confirm infection with A. phagocytophilum. PCRs were prepared using
GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA). Amplification
was completed using the following PCR cycling conditions: 1 denaturing
cycle for 3 min at 95 °C, followed by 34 cycles of 1-min denaturation
at 95 °C, 1 min at the annealing temperature ([67]Table 1) and an
extension of 72 °C for 30 s. A final extension step of 5 min at 72 °C
was performed. Predicted product sizes are displayed in [68]Table 1.
PCR product sizes were confirmed by gel electrophoresis and compared
with a 100 bp ladder (NEB, Ipswich, MA, USA). Gels were visualized with
an iBright FL1500 Imaging System (Thermo Scientific, Whaltham, MA, USA)
to confirm the infection of mice ([69]Figure S1a).
Table 1.
Primers used for PCR and qRT-PCR amplifications.
Primer Name Primer Sequence Ta * Product Size (bp) Reference
Mactin F 5′-ACGCAGAGGGAAATCGTCCGTGAC-3′ 60 °C 101 [[70]24]
Mactin R 5′-ACGCGGGAGGAAGAGGATGCGGCAGTG-3′ 60 °C
Actin Is F 5′-GGTCATCACAATCGGCAA-3′ 54 °C 108 [[71]25]
Actin Is R 5′-ATGGAGTTGTACGTGGTCTC-3′ 54 °C
P44 F 5′-ATGGAAGGTAGTGTTGGTTATGGTATT-3′ 56 °C 77 [[72]26]
P44 R 5′-TTGGTCTTGAAGCGCTCGTA-3′ 56 °C
16s rRNA F 5′-GGTGAGTAATGCATAGGAATC-3′ 53 °C 108 [[73]27]
16s rRNA R 5′-GCTCATCTAATAGCGATAAATC-3′ 53 °C
rpoB F 5′-CTTTATCCTGCTTTAGAACAACATC-3′ 52 °C 286 [[74]18]
rpoB R 5′-GGTCCGTATGGTCTGGTTACT-3′ 52 °C
Ifng F 5′-AGCGTCATTGAATCACACCT-3′ 54 °C 196 This study
Ifng R 5′-ATCAGCAGCGACTCCTTTTC-3′ 54 °C
IL1βF 5′-CCTGTGTAATGAAAGACGGC-3′ 54 °C 216 This study
IL1βR 5′-TGTCCTGACCACTGTTGTTT-3′ 54 °C
Irf1 F 5′-ATAACTCCAGCACTGTCACC-3′ 54 °C 177 This study
IrF1 R 5′-AAGGTCTTCGGCTATCTTCC-3′ 54 °C
Stat2 F 5′- TGGGACTTCGGCTTCTTGAC-3′ 57 °C 247 This study
Stat2 R 5′- TCTTGGGATTTGGGCTGAGC-3′ 57 °C
S100a8 F 5′-CACCATGCCCTCTACAAGAA-3′ 54 °C 161 This study
S100a8 R 5′-CCCACTTTTATCACCATCGC-3′ 54 °C
Acan F 5′-CAGATGGCACCCTCCGATAC-3′ 57 °C 151 This study
Acan R 5′-GACACACCTCGGAAGCAGAA-3′ 57 °C
Matn3 F 5′-GAGGGTGGCTGTGGTGAACT-3′ 59 °C 160 This study
Matn3 R 5′-GGCTTCCTCCATCGCTGTCT-3′ 59 °C
[75]Open in a new tab
* Annealing temperatures.
2.3. Tick Infestations
For A. phagocytophilum tick infections, 200 larval I. scapularis ticks
were placed on mice after confirming infection in the blood ([76]Figure
1a). Mice were separated into individual mesh bottom cages placed above
a water trap to collect the fed ticks. Mice were anesthetized for 30
min with 1.25% to 2% isoflurane to allow the larvae to attach. Engorged
ticks were collected from the water baths after 3, 4 and 5 days of
feeding ([77]Figure 1a). The mice were euthanized with CO[2], followed
by cervical fracture and heart puncture exsanguination. The collected
ticks were washed in 2% bleach and autoclaved water, placed into groups
of 25, and allowed to molt into nymphs.
DNA was purified from 5 pooled nymphs from each group of infected and
control ticks to confirm infection or lack of. Ticks were placed at −80
°C for 1 h and DNA was isolated using the Quick-DNA/RNA Miniprep kit
(Zymo, Irvine, CA, USA), according to the manufacturer’s instructions.
DNA quantity and quality were tested as described above, and a PCR on
the I. scapularis actin gene was performed to determine the presence of
PCR inhibitors. Anaplasma phagocytophilum infection (or lack of) was
confirmed by PCR amplification of the rpoB and p44 genes ([78]Table 1;
[79]Figure 1a). Amplification of the PCR products was carried out using
similar cycling conditions as described above. Predicted product sizes
are displayed in [80]Table 1. Positive and negative PCRs were confirmed
by gel electrophoresis as described above. Additionally, the relative
levels of bacterial infection were assessed by qPCR, using the ΔCt
value of A. phagocytophilum p44 normalized by tick actin with the
following formula:
[MATH:
ΔCt=2−(ct A<
/mi>naplasma p44−ct tick actin) :MATH]
qPCRs were performed using PowerUp™ SYBR™ Green Master Mix (Applied
Biosystems, Whaltham, MA, USA), using same primers as for PCR analysis
([81]Table 1). Amplification, melt curves, and data were analyzed with
CFX Maestro Software (Bio-Rad, Hercules, CA, USA).
Due to the potential effect of the mutation of C3H/HeJ mice tlr4 in the
local immune responses to A. phagocytophilum transmission, we decided
to use a different mouse strain. The C57BL/6J mouse strain has
previously been used for the study of murine systemic immune responses
during A. phagocytophilum infection and the role of IFN-γ/STAT1
[[82]11], therefore, we used this same strain to define local immune
responses to bacterial transmission. A. phagocytophilum-infected and
-uninfected nymphs were used to infest 6 week old C57BL/6J male mice
(The Jackson Laboratory Bar Harbor, ME, USA). Twenty-five I. scapularis
nymphs were placed on each mouse. Mice were anesthetized as previously
described for the larvae ([83]Figure 1b). The ticks were allowed to
feed for 3 days to allow 24 h post-transmission of A. phagocytophilum
[[84]28]. After this time, the mice were euthanized with CO₂, followed
by cervical fracture. Three (3) mm skin biopsies were taken of the bite
sites, utilizing Integra disposable biopsy punches (Militex,
Saint-Pries, France) for RNAseq, and 5 mm skin biopsies were taken for
qRT-PCR ([85]Figure 1b). Skin samples far from where the ticks were
located were taken to determine the gene expression in intact skin
(baseline). The partially engorged nymphs were collected for excision
of their midguts and salivary glands. The skin was placed in 500 μL
RNALater (Invitrogen, Carlsbad, CA, USA).
2.4. RNA-Seq and Pathway Analysis of Skin Biopsies
RNA extraction, library preparations, sequencing reactions and
bioinformatic analysis were conducted at GENEWIZ, LLC. (South
Plainfield, NJ, USA) as follows: total RNA was extracted using Qiagen
RNeasy Plus Universal mini kit following the manufacturer’s
instructions (Qiagen, Hilden, Germany). The quantity and quality of the
RNA samples were assessed using a Qubit 2.0 Fluorometer (Life
Technologies, Carlsbad, CA, USA) and an Agilent TapeStation 4200
(Agilent Technologies, Palo Alto, CA, USA), respectively.
The RNA libraries were prepared using the NEBNext Ultra II RNA Library
Prep Kit (NEB, Ipswich, MA, USA), following the manufacturer’s
instructions for Illumina. Briefly, mRNAs were first enriched with
Oligo(dT) beads, followed by fragmentation for 15 min at 94 °C and cDNA
synthesis. cDNA was adenylated at 3′ends and end repaired. Universal
adapters were ligated to cDNA fragments, followed by index addition and
library enrichment by limited-cycle PCR. The quality of the libraries
was validated on the Agilent TapeStation (Agilent Technologies, Palo
Alto, CA, USA). Libraries were quantified with a Qubit 2.0 Fluorometer
(Invitrogen, Carlsbad, CA, USA) and quantitative PCR (KAPA Biosystems,
Wilmington, MA, USA).
The libraries were sequenced on an Illumina HiSeq instrument (4000)
according to the manufacturer’s instructions, using a 2 × 150 bp paired
end (PE) configuration. HiSeq Control Software (HCS) v2.0.12 was used
for image analysis and base calling. After investigating the quality of
the raw data, possible adapter sequences and nucleotides with poor
quality were trimmed. The trimmed reads were mapped to the reference
genome, using STAR aligner v.2.5.2b. Unique gene hit counts were
calculated using featureCounts from Subread package v.1.5.2. Only
unique reads that fell within exon regions were counted. The complete
sequencing data were deposited on NCBI, and accession numbers can be
found in the data availability section.
Upregulated and downregulated genes with adjusted p-value (padj) < 0.05
and log2 fold changes of 1 or more (−1 or less for downregulated genes)
were used to identify pathway over-representation using Reactome
([86]https://reactome.org/, accessed on 19 February 2022). Only
pathways with p < 0.05 and false discovery rate (FDR) < 0.05 were
considered as over-represented.
2.5. qRT-PCR of Skin Biopsies
RNA was extracted from the skin using TRIZOL (Invitrogen, Whaltham, MA,
USA) according to the manufacturer’s specifications with small
modifications. Briefly, the RNALater was washed off the tissues with 1×
phosphate buffered saline (PBS), and skin samples were quickly flash
froze with LN[2]. The frozen tissue was homogenized with a mortar and
pestle. One (1) mL of TRIZOL was added to the tissue. The aqueous phase
was taken and mixed 1:1 with 70% ethanol. RNA was then isolated using
PureLink™ RNA Mini Kit (Ambion, Carlsbad, CA, USA), according to the
manufacturer’s indications. RNA was quantified and quality was assessed
using a NanoQuant Infinite M200 Pro (Tecan, Switzerland). RNA (100 ng)
was used to synthesize cDNA with the Verso cDNA Synthesis Kit (Thermo
Scientific, Whaltham, MA, USA). qPCRs were performed using PowerUp™
SYBR™ Green Master Mix (Applied Biosystems, Whaltham, MA, USA).
Interferon gamma (Ifn-g), interleukin 1β (Il1b), interferon regulatory
factor 1 (Irf1), S100 calcium binding protein A8 (S100a8), Aggrecan
(Acan) and Matrilin 3 (matn3) were amplified using the primers
described in [87]Table 1. The amplifications were performed in a CFX
Opus 96 Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA).
Amplification, melt curves, and data were analyzed with CFX Maestro
Software (Bio-Rad, Hercules, CA, USA). The relative differences were
calculated using the ΔΔCt method [[88]29] as follows:
[MATH:
ΔΔCT=<
/mo>(2−
(Ct gene of i
nterest<
mo> treatment−Ct actin t
reatment
))(2−(Ct gene of interest intact
skin−Ct<
mo> actin intact skin)) :MATH]
The expression of the genes was normalized to mouse actin ([89]Table
1). The expression of immune genes in skin samples at the bite site of
uninfected and infected ticks was normalized to that of the intact skin
(baselines). Statistical differences in gene expression between
conditions was evaluated using an unpaired two-tailed t-test with
GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA, USA). Outliers
were determined using GraphPad 9.3.1.
The specificity of the primers was confirmed by RT-PCR of RNA isolated
from mouse hearts followed by Sanger sequencing (Etonbio, Union, NJ,
USA). The forward and reverse sequences were assembled with Geneious
Prime (Biomatters, Inc., San Diego, CA, USA) and identity was confirmed
through BLAST (NCBI). Accession numbers for the sequences amplified by
the primers used here in can be found in the data availability
statement.
3. Results
3.1. Tick Feeding Induces the Expression of Neutrophil Chemotaxis and
Inflammatory Responses in the Skin
Pathogen-free certified larvae fed upon naïve mice until repletion.
Following molting, the absence of A. phagocytophilum infection in
control nymphs was confirmed by DNA extraction, followed by negative
amplification of p44 and RpoB genes from A. phagocytophilum ([90]Figure
S1b). Uninfected (control) nymphs fed upon C57BL/6J for 3 days, and 2
mm skin biopsy punches were taken from the bite site. RNAseq and
differential gene expression (DEG) analyses were performed on RNA
isolated from the skin samples. A total of 1213 genes were
differentially regulated upon tick feeding, when compared with the
intact skin ([91]Table 2). A total of 797 genes were upregulated and
416 were downregulated during tick feeding. Principal component
analysis (PCA) demonstrated a clear separation of the transcriptomic
profiles between the intact skin and uninfected tick bite sites
([92]Figure 2a). Volcano plot analysis of intact skin samples versus
uninfected tick bite sites showed the upregulation of several
immune-related genes (including genes encoding chemokines and
chemoattractant Saa3), and the downregulation of iron homeostasis genes
(such as Hamp2) and transcription factors (such as RorC) ([93]Figure
2b).
Table 2.
Significantly differentially regulated genes affected by tick feeding
and Anaplasma phagocytophilum transmission.
Comparison Upregulated Genes Downregulated Genes Total Significantly
DEGs
Intact skin v uninfected tick bite sites 797 416 1213
Intact skin v Anaplasma-infected tick bite 1417 1142 2559
Uninfected tick bite sites v Anaplasma-infected tick bite 476 146 622
[94]Open in a new tab
Figure 2.
[95]Figure 2
[96]Open in a new tab
Transcriptional profile of immune genes during tick feeding. (a)
Principal component analysis (PCA) of the transcriptional profiles of
intact skin samples (baseline = pink dots) versus uninfected control
ticks (blue dots). (b) The global transcriptional change across intact
skin (baseline) and skin samples collected from uninfected tick bite
sites (control) was visualized by a volcano plot. Each data point in
the scatter plot represents a gene. The log2 fold change of each gene
is represented on the x-axis and the log10 of its adjusted p-value is
on the y-axis. Genes with an adjusted p-value less than 0.05 and a log2
fold change greater than 1 represent upregulated genes (red dots).
Genes with an adjusted p-value less than 0.05 and a log2 fold change
less than −1 are indicated by blue dots (downregulated genes). (c) A
bi-clustering heatmap was used to visualize the expression profile of
the top 30 differentially expressed genes sorted by their adjusted
p-value by plotting their log2 transformed expression values in
samples. The pink squares at the top represent “control” uninfected
ticks, whereas the intact skin (baseline) is shown in light blue.
Heatmap analysis of the 30 most differentially regulated genes resulted
in five gene clusters of co-regulated gene ([97]Figure 2c). Cluster 1
included keratine-encoding genes important in epithelial cell integrity
in the skin (krt16 and krt6b); enzymes involved in phospholipid and
heme metabolism, and glycolysis (Plbd1, Hmox1, and Eno1); and to
signaling proteins, including Arrdc3 that allows G protein signaling,
and Saa3. Cluster 2 comprised several lectins, such as Chil3, Sell, and
Clec4d; the glycoprotein Cd300lf; and 2610528a11Rik, a gene encoding G
protein-coupled receptor 15 ligand induced during several inflammatory
conditions in the skin [[98]30]. Cluster 3 contained a gene without
known function (Gm45819) and a circadian transcriptional repressor
(Cry1). Cluster 4 was formed by two chemokines, one involved in
macrophage attraction (Ccl7), and Ccl2 that is a chemoattractant for
monocytes and basophils. It also contained a lipocalin (Lcn2), a
metalloproteases inhibitor (Timp1), and a proteoglycan (Prg4). Cluster
5 contained two genes involved in chromosome segregation (Kif22 and
Nuf2), immune response associated genes (Slfn4, S100a9, S100a8, and
Il1b), epidermal maintenance and development (Stfa3 and Stfa1), and
other genes (Slc15a3 and Adamts4). All clusters showed upregulation of
the co-regulated genes, with the exception of Cluster 3 that showed
higher levels of expression in the intact skin (baseline; [99]Figure
2c). Enrichment analysis based on gene ontology (GO) ([100]Figure S2)
showed an over-representation of genes associated with immune
responses, neutrophil chemotaxis, cell division, and chromosome
segregation. Pathway enrichment (PE) analysis ([101]Table S1) of
upregulated genes demonstrated enrichment of interleukin-10 signaling
associated genes, neutrophil degranulation, Th2 signaling, and mitotic
spindle, potentially indicating the division of keratynocytes in
response to the inflammation and damage associated with the tick bite
as previously reported in mouse biopsies [[102]25]. Downregulated genes
did not show any significant enrichment in pathways ([103]Table S2).
3.2. Anaplasma Phagocytophilum Transmission Induces the Upregulation of
Interferon Signaling Genes
Ixodes scapularis larvae fed upon A. phagocytophilum PCR-positive mice
([104]Figure S1a). Ticks were allowed to molt and DNA was extracted
from five nymphs from each group to confirm infection status. Anaplasma
phagocytophilum-positive nymphs ([105]Figure S1b) with varying relative
levels of bacterial infection ([106]Figure S1c) were infested onto
naïve C57BL/6J mice for 3 days. Skin samples were taken from the bite
site and RNA was isolated for RNAseq analysis. DEG analysis identified
a total of 2559 DEGs between the intact skin and skin samples from the
bite site of Anaplasma phagocytophilum-infected ticks, including 1417
upregulated and 1142 downregulated genes. Comparatively, 622 DEGs were
identified when uninfected tick and infected tick bite sites were
compared. This encompassed 476 upregulated and 146 downregulated genes,
indicating a synergistic effect between A. phagocytophilum and the tick
([107]Table 2). PCA showed the separation of control samples and
samples from the bite site of A. phagocytophilum-infected ticks and of
uninfected ticks. Similar separation was observed between intact skin
and infected ticks’ bite sites. In both cases, the presence of one
outlier from the A. phagocytophilum ticks was detected ([108]Figure 3a
and [109]Figure S3a). Nevertheless, this sample showed similar
co-regulatory gene expression as the other samples ([110]Figure 3c and
[111]Figure S3a). Anaplasma4, on the other hand, showed a slightly
lower upregulation of immune genes when compared with skin samples from
bite sites of uninfected ticks and intact skin ([112]Figure 3c and
[113]Figure S3c; [114]Supplementary Files S1 and S2). This might be due
to lower levels of infection in the tick or due to a delayed attachment
of the tick leading to less time for A. phagocytophilum transmission,
although this is speculative since we did not test bacterial numbers in
the skin.
Figure 3.
[115]Figure 3
[116]Open in a new tab
Transmission of Anaplasma phagocytophilum leads to Th1 and interferon
signaling induction. (a) Principal component analysis (PCA) of the
transcriptional profiles of skin samples from uninfected tick bite
sites (control; pink dots) versus skin samples from infected tick bite
sites (Anaplasma; blue dots). (b) The global transcriptional change
across the skin samples collected from uninfected (control) and
infected tick bite sites (Anaplasma) was visualized by a volcano plot.
Each data point in the scatter plot represents a gene. The log2 fold
change of each gene is represented on the x-axis and the log10 of its
adjusted p-value is on the y-axis. Genes with an adjusted p-value less
than 0.05 and a log2 fold change greater than 1 (upregulated) are
indicated by red dots. Genes with an adjusted p-value less than 0.05
and a log2 fold change less than −1 (downregulated) are indicated by
blue dots. (c) A bi-clustering heatmap was used to visualize the
expression profile of the top 30 differentially expressed genes sorted
by their adjusted p-value by plotting their log2 transformed expression
values in samples. The pink squares at the top represent infected tick
bite sites (Anaplasma). The light blue squares are the skin samples
from uninfected tick’s bite sites (control).
Analysis of the gene expression using volcano plots showed several
immune-related genes that are significantly upregulated during
transmission of A. phagocytophilum when compared with uninfected ticks’
bite sites ([117]Figure 3b), including the gene encoding tumor necrosis
factor (tnf), and chemokines ccl4 and cxcl10, which are attractants of
macrophages, natural killer cells, and T cells. Interestingly, genes
involved in extracellular matrix formation were downregulated; for
example, the gene encoding matn3. Heatmap analysis identified six
clusters of co-regulated genes ([118]Figure 3c). The first cluster
upregulated in Anaplasma transmission skin samples, included the genes
encoding for the interferon gamma inducible GTPase Ifgga3 protein
(Gm4841), lymphocyte antigen 6c2 (Ly6c2; which acts as an acetylcholine
receptor), the endoplasmic reticulum/Golgi membrane-spanning 4-domains
subfamily A, member 4C (Ms4a4c), and the ubiquitin-like modifier
protein ISG15 (Isg15). The second cluster contained two
interferon-induced genes (Ifi213 and Oas3), regulators of Th1 cytokine
secretion (Phf11b and Phf11d), and an enzyme involved in arginine
biosynthesis (Ass1), which showed upregulation in the skin samples from
infected ticks bite sites.
A third cluster of upregulated genes involved Slfn4, the gene encoding
an interferon regulatory factor 7 (Irf7), and the interferon-induced
gene Oasl2. Two genes encoding G receptor interacting proteins were
also found in this group (Rgs5 and Slc9a3r1). The fourth cluster only
contained two genes slightly upregulated during A. phagocytophilum
transmission: an interferon activated gene (Ifi214) and a pseudogene
(Gm654). The fifth cluster contained the highest number of genes and
included several immune-related genes (Gzmb, Gm12185, Trim30b, Ccl4,
and Tnf) and two lipid transport proteins (Apol9b and Apol9a). The last
cluster included genes encoding two glycoproteins (Cd300lf and Clec4e)
and an interferon-induced protein (Ifi44), which are involved in innate
immune responses ([119]Figure 3c). Several of the immune-related
clusters upregulated in A. phagocytophilum transmission sites versus
controls were also observed when compared with intact skin (baseline;
[120]Figure S3c).
Gene ontology (GO) enrichment analysis of the differentially regulated
genes during A. phagocytophilum transmission identified the enrichment
of genes involved in the cellular response to interferon β (Ifn-β) and
Ifn-γ ([121]Figure 4). Enrichment of interferon signaling was also
detected using PE analysis of the significantly upregulated genes
([122]Table 3 and [123]Table S3). Interestingly, antiviral mechanisms
also showed enrichment in GO and pathway analysis, likely due to the
intracellular nature of A. phagocytophilum infection. Other interleukin
signaling pathways (IL-1, IL-10, and IL-12) were also observed in the
enrichment analysis. Th2 responses were enriched to a lesser degree and
likely represented the responses to the tick feeding, as these pathways
were significantly over-represented in uninfected tick bite sites,
unlike interferon signaling that was not found to be upregulated during
uninfected tick feeding ([124]Table S1). This suggests that A.
phagocytophilum transmission leads to the activation of Th1 signaling
pathways in the skin.
Figure 4.
[125]Figure 4
[126]Open in a new tab
Gene ontology (GO) analysis of genes differentially expressed during
transmission of Anaplasma phagocytophilum.
Table 3.
Pathway enrichment (PE) analysis of upregulated genes during
transmission of Anaplasma phagocytophilum.
Pathway Identifier Pathway Name Number
Entities Found Number
Entities
Total Entities
p-Value Entities FDR
R-HSA-909733 Interferon alpha/beta signaling 50 188 1.11 × 10^−16 1.52
× 10^−14
R-HSA-6783783 Interleukin-10 signaling 38 86 1.11 × 10^−16 1.52 ×
10^−14
R-HSA-913531 Interferon signaling 81 394 1.11 × 10^−16 1.52 × 10^−14
R-HSA-1280215 Cytokine signaling in immune system 149 1092 1.11 ×
10^−16 1.52 × 10^−14
R-HSA-168256 Immune system 213 2684 1.11 × 10^−16 1.52 × 10^−14
R-HSA-449147 Signaling by interleukins 70 643 4.66 × 10^−15 5.32 ×
10^−13
R-HSA-877300 Interferon gamma signaling 41 250 1.18 × 10^−14 1.15 ×
10^−12
R-HSA-380108 Chemokine receptors bind chemokines 17 57 1.40 × 10^−10
1.21 × 10^−8
R-HSA-6785807 Interleukin-4 and interleukin-13 signaling 26 211 2.48 ×
10^−7 1.88 × 10^−5
R-HSA-1169410 Antiviral mechanism by IFN-stimulated genes 14 94 1.97 ×
10^−5 0.001342146
R-HSA-375276 Peptide ligand-binding receptors 20 203 1.29 × 10^−4
0.007973888
R-HSA-1169408 ISG15 antiviral mechanism 11 83 3.99 × 10^−4 0.022716167
R-HSA-9705462 Inactivation of CSF3 (G-CSF) signaling 6 27 6.44 × 10^−4
0.03349729
[127]Open in a new tab
Pathway enrichment (PE) analysis of downregulated genes during A.
phagocytophilum showed an over-representation of pathways involved in
ECM integrity, including ECM organization, fibrils formation, and
several collagen organization enzymes ([128]Table 4). Some genes
involved in these pathways were downregulated in uninfected tick bite
sites ([129]Figure S2). Nevertheless, pathways involved in ECM
integrity were not over-represented in uninfected tick bite sites
([130]Table S2). Curiously, although the upregulation of interferon and
interleukin signaling pathways were detected during A. phagocytophilum
transmission when compared with intact skin ([131]Table S3), the ECM
organization pathways were not over-represented in downregulated genes
([132]Table S4).
Table 4.
Pathway enrichment (PE) analysis of downregulated genes during
transmission of Anaplasma phagocytophilum.
Pathway Identifier Pathway Name Number
Entities Found Number
Entities
Total Entities
p-Value Entities FDR
R-HSA-1474244 Extracellular matrix organization 29 329 1.11 × 10^−16
3.26 × 10^−14
R-HSA-2022090 Assembly of collagen fibrils and other multimeric
structures 13 67 7.42 × 10^−13 5.98 × 10^−11
R-HSA-1474290 Collagen formation 15 104 8.05 × 10^−13 5.98 × 10^−11
R-HSA-1474228 Degradation of the extracellular matrix 17 148 8.20 ×
10^−13 5.98 × 10^−11
R-HSA-8948216 Collagen chain trimerization 11 44 3.13 × 10^−12 1.72 ×
10^−10
R-HSA-1650814 Collagen biosynthesis and modifying enzymes 13 76 3.52 ×
10^−12 1.72 × 10^−10
R-HSA-3000178 13 79 5.66 × 10^−12 2.38 × 10^−10
R-HSA-216083 Integrin cell surface interactions 11 86 3.39 × 10^−9 1.22
× 10^−7
R-HSA-1442490 Collagen degradation 10 69 5.56 × 10^−9 1.78 × 10^−7
R-HSA-419037 NCAM1 interactions 6 44 9.59 × 10^−6 2.78 × 10^−4
R-HSA-1566948 Elastic fiber formation 6 46 1.23 × 10^−5 3.20 × 10^−4
R-HSA-8874081 MET activates PTK2 signaling 5 32 2.86 × 10^−5 6.86 ×
10^−4
R-HSA-3000171 Non-integrin membrane-ECM interactions 6 61 5.86 × 10^−5
0.001289879
R-HSA-375165 NCAM signaling for neurite outgrowth 6 70 1.24 × 10^−4
0.002108039
R-HSA-186797 Signaling by PDGF 6 70 1.24 × 10^−4 0.002108039
R-HSA-3656244 Defective B4GALT1 causes B4GALT1-CDG (CDG-2d) 3 9 1.38 ×
10^−4 0.002108039
R-HSA-3656225 Defective CHST6 causes MCDC1 3 9 1.38 × 10^−4 0.002108039
R-HSA-3656243 Defective ST3GAL3 causes MCT12 and EIEE15 3 9 1.38 ×
10^−4 0.002108039
R-HSA-8875878 MET promotes cell motility 5 45 1.41 × 10^−4 0.002108039
R-HSA-2022854 Keratan sulfate biosynthesis 4 37 7.38 × 10^−4
0.010327613
R-HSA-2129379 Molecules associated with elastic fibers 4 38 8.14 ×
10^−4 0.011395335
R-HSA-2022857 Keratan sulfate degradation 3 22 0.001819501 0.023653508
R-HSA-2243919 Crosslinking of collagen fibrils 3 24 0.002325531
0.027906369
R-HSA-1638074 Keratan sulfate/keratin metabolism 4 52 0.002542129
0.029455221
R-HSA-399710 Activation of AMPA receptors 2 7 0.002677747 0.029455221
R-HSA-6806834 Signaling by MET 5 88 0.002781469 0.03059616
R-HSA-1369062 ABC transporters in lipid homeostasis 3 29 0.00394569
0.039456898
R-HSA-8951671 RUNX3 regulates YAP1-mediated transcription 2 9
0.004364679 0.043646794
[133]Open in a new tab
3.3. Differentially Expressed Genes (DEGs) Stimulated during Tick Feeding and
A. Phagocytophilum Transmission
To determine genes and pathways that are affected by both the tick
feeding and A. phagocytophilum transmission, the DEGs that are
upregulated and downregulated during the bite of uninfected and
infected ticks, when compared with intact skin expression, were
identified ([134]Supplemental File S7). Pathway enrichment (PE) of
genes upregulated in both conditions showed an over-representation of
interleukin-10 signaling, neutrophil degranulation, chemokine
signaling, Th2 cytokine signaling, and other pathways enriched during
tick feeding compared with intact skin expression ([135]Table 5 and
[136]Table S1), corroborating that the enrichment of these pathways
during A. phagocytophilum transmission is the result of the synergetic
effect of the tick and bacterial transmission. By comparison, PE
analysis of genes upregulated during transmission of A. phagocytophilum
solely identified an over-representation of interferon signaling genes,
interleukin-10 signaling, and other immune response pathways
([137]Table S5), confirming the effect of the bacterial transmission on
these signaling pathways. In the case of the DEGs identified as
upregulated during the tick bite only, no significant enrichment of
pathways was detected ([138]Table S6).
Table 5.
Pathway enrichment (PE) analysis of upregulated genes shared between
skin samples from uninfected tick bite sites and infected tick bite
sites when compared with intact skin samples.
Pathway Identifier Pathway Name Number
Entities Found Number
Entities Total Entities
p-Value Entities FDR
R-HSA-6783783 Interleukin-10 signaling 36 86 7.12 × 10^−14 1.17 ×
10^−10
R-HSA-6798695 Neutrophil degranulation 61 480 4.41 × 10^−13 3.62 ×
10^−10
R-HSA-380108 Chemokine receptors bind chemokines 24 57 7.27 × 10^−11
3.97 × 10^−8
R-HSA-6785807 Interleukin-4 and interleukin-13 signaling 42 211 2.18 ×
10^−8 8.94 × 10^−6
R-HSA-2500257 Resolution of sister chromatid cohesion 28 134 4.57 ×
10^−7 1.50 × 10^−4
R-HSA-2467813 Separation of sister chromatids 27 195 1.10 × 10^−6 3.00
× 10^−4
R-HSA-141424 Amplification of signal from the kinetochores 22 94 7.82 ×
10^−6 0.001602622
R-HSA-141444 Amplification of signal from unattached kinetochores via a
MAD2 inhibitory signal 22 94 7.82 × 10^−6 0.001602622
R-HSA-5663220 RHO GTPases activate formins 24 149 1.20 × 10^−5
0.002177163
R-HSA-68877 Mitotic prometaphase 31 211 1.79 × 10^−5 0.002933421
R-HSA-9648025 EML4 and NUDC in mitotic spindle formation 24 121 3.02 ×
10^−5 0.004498095
R-HSA-69618 Mitotic spindle checkpoint 22 111 1.34 × 10^−4 0.018203955
[139]Open in a new tab
In the case of shared downregulated genes, no significant PE was
observed ([140]Table 6), suggesting that the transmission of A.
phagocytophilum leads to the downregulation of distinct pathways when
compared with tick feeding. The analysis of genes downregulated only
during A. phagocytophilum transmission, identified pathways involved in
muscle contraction, collagen related pathways, and extracellular matrix
organization ([141]Table S7). As in the case of shared downregulated
genes, the PE analysis of genes downregulated during uninfected tick
feeding did not identify any significantly over-represented pathways
([142]Table S8). These results were similar to the analysis of all DEGs
downregulated during tick feeding versus expression in intact skin
([143]Table S2). This confirmed that A. phagocytophilum transmission,
and not the tick feeding, is responsible for the detected effects on
ECM organization. Whether this effect is due to bacterial manipulation
of cells in the skin or due to changes in tick saliva during infection
with A. phagocytophilum remains to be determined.
Table 6.
Pathway enrichment (PE) analysis of downregulated genes shared between
skin samples from uninfected tick bite sites and infected tick bite
sites when compared with intact skin samples.
Pathway Identifier Pathway Name Number Entities Found Number Entities
Total Entities
p-Value Entities FDR
R-HSA-400253 Circadian clock 9 105 0.001723 0.60096047
R-HSA-5682910 LGI-ADAM interactions 3 14 0.005775 0.60096047
R-HSA-3000480 Scavenging by Class A receptors 5 49 0.008965 0.60096047
R-HSA-8874081 MET activates PTK2 signaling 4 32 0.009601 0.60096047
R-HSA-3000178 ECM proteoglycans 6 79 0.016775 0.60096047
R-HSA-2022870 Chondroitin sulfate biosynthesis 3 25 0.026832 0.60096047
R-HSA-1482922 Acyl chain remodeling of PI 3 25 0.026832 0.60096047
R-HSA-391903 Eicosanoid ligand-binding receptors 3 25 0.026832
0.60096047
R-HSA-8949275 RUNX3 regulates immune response and cell migration 2 10
0.02741 0.60096047
R-HSA-8875878 MET promotes cell motility 4 45 0.029135 0.60096047
R-HSA-1482925 Acyl chain remodeling of PG 3 26 0.029633 0.60096047
R-HSA-1442490 Collagen degradation 5 69 0.033148 0.60096047
R-HSA-430116 GP1b-IX-V activation signaling 2 12 0.038188 0.60096047
R-HSA-3000170 Syndecan interactions 3 29 0.038924 0.60096047
R-HSA-1482801 Acyl chain remodeling of PS 3 31 0.045843 0.60096047
R-HSA-6785807 Interleukin-4 and interleukin-13 signaling 10 211
0.046493 0.60096047
R-HSA-1650814 Collagen biosynthesis and modifying enzymes 5 76 0.046724
0.60096047
R-HSA-391908 Prostanoid ligand receptors 2 14 0.050296 0.60096047
R-HSA-2214320 Anchoring fibril formation 2 15 0.0568 0.60096047
[144]Open in a new tab
3.4. Confirmation of Th1 Cytokines Upregulation and Downregulation of ECM
Genes by qRT-PCR
The expression patterns of selected genes identified by RNAseq were
confirmed by qRT-PCR. RNA was isolated from skin biopsies of the bite
site of uninfected and A. phagocytophilum ticks and were normalized to
the gene expression of intact skin from the same mice. Similar to the
tick batches used for the infestation of mice during the RNAseq
experiments, relative bacterial levels were highly variable
([145]Figure S1d). The upregulation of Ifn-γ, stat2, and il1β during A.
phagocytophilum transmission were validated ([146]Figure 5a–c).
Although Irf1 was slightly upregulated in the bite site of A.
phagocytophilum ticks, this difference was not statistically
significant ([147]Figure 5d). S100a8, Acan, and matn3 were upregulated
in samples taken from uninfected tick bite sites ([148]Figure 5e–g).
Acan and matn3 encode for proteins with roles in ECM integrity; thus,
validating the upregulation of interferon and interleukin-1 mediated
signaling related genes and the downregulation of genes involved in ECM
integrity. Interestingly, although the relative expression of immune
genes (Infg, stat2, and il1β) was significantly lower in intact skin
when compared with the bite site of A. phagocytophilum-infected ticks,
the expression of Acan and matn3 was not significantly different
([149]Figure S4), thus, confirming that expression of these ECM
integrity genes is similar in intact skin and A. phagocytophilum
transmission sites.
Figure 5.
[150]Figure 5
[151]Open in a new tab
qRT-PCR confirmation of DEGs in skin samples taken from the bite site
of uninfected and Anaplasma phagocytophilum-infected ticks. The ΔΔCt
method was used to calculate the fold change gene expression on skin
samples from uninfected tick bite sites (control; blue) versus A.
phagocytophilum-infected (Anaplasma; red). Gene expression levels were
normalized to actin and divided by normalized gene expression in intact
skin. The expression of: (a) interferon γ (Ifn-γ; ** p = 0.0084); (b)
signal transducer and activator of transcription 2 (stat2; * p =
0.0132); (c) interleukin β (Il1β; *** p = 0.0005); (d) interferon
regulatory factor 1 (Irf1; ns: not significant); (e) S100 calcium
binding protein A8 (S100a8; * p = 0.0412); (f) Aggrecan (Acan; * p =
0.0352); and (g) matn3 (matn3; ** p = 0.0091). Each dot in the graph
represents a single skin sample. The height of the bar and the lines
symbolize the average and the standard mean of the error (SEM),
respectively. The graphs are representative from three independent
experiments.
4. Discussion
Initial responses within the skin can ultimately define the outcome of
infection by a vector-borne pathogen. Skin cells, including immune
cells, keratinocytes, and endothelial cells, serve as the site of
initial replication for vector-borne viruses, bacteria, and parasites
[[152]31,[153]32,[154]33,[155]34,[156]35]. Arthropod inoculation of
some viruses can lead to increased severity of pathologies associated
with infection [[157]31]. Further, arthropod saliva influences
responses beyond the skin. For example, inoculation of mosquito
salivary gland extracts (SGE) enhanced the migration of neutrophils and
dendritic cells into draining lymph nodes and boosted the pathogenesis
of the virus during antibody-dependent enhancement of dengue [[158]34].
The role of single salivary proteins in the stimulation of immune cells
and the enhanced pathogenesis of disease has also been investigated in
mosquito models. In Zika virus (ZIKV), neutrophil-stimulating factor 1
(NeSt1) activates neutrophils and leads to augmented early virus
replication [[159]36]. Similarly, proteins within Lutzomia longipalpis
saliva affect neutrophil function, inducing macrophage migration into
the bite site and increasing Leishmania chagasi and Leishmania infantum
replication [[160]32,[161]33,[162]35]. Thus, arthropod saliva and other
vector-derived factors are important determinants of pathogenesis of
vector-borne pathogens.
In the case of tick-borne pathogens, little is known of the cellular
and immune factors that influence their establishment and how tick
saliva and tick modulatory molecules affect pathogen replication and
pathogenesis [[163]37]. In the case of Rickettsia parkeri, although
tick saliva and tick feeding increase skin pathology during bacterial
infection, bacterial replication at the inoculation site was reduced in
the presence of tick salivary components [[164]38,[165]39]. As in the
case of mosquitoes, Amblyomma maculatum feeding resulted in variable
infiltration of macrophages and neutrophils in rhesus monkeys’ skin,
whereas bacterial inoculation at the tick feeding site led to marked
neutrophil and macrophage translocation at the dermis [[166]39]. Ixodes
scapularis feeding also led to a significant increase in the number of
neutrophils and macrophages in murine skin [[167]25], which was
consistent with previous transcriptional analysis of tick bite sites in
murine models [[168]40] and our results ([169]Figure S2). According to
our results, genes involved in neutrophil chemotaxis and degranulation
were induced during I. scapularis feeding and A. phagocytophilum
transmission ([170]Figure 3 and [171]Figure S5). This includes the
upregulation of CXCL1 and CXCL2 and the receptors CCR5, CCR7, and CCRL2
([172]Supplementary File S8), which are involved in the activation,
migration, effector, and antigen presentation functions of neutrophils
[[173]41,[174]42]. Pathways enrichment (PE) analysis of genes
upregulated during tick feeding and A. phagocytophilum transmission
when compared with intact skin showed that neutrophil degranulation
pathways are enriched in both ([175]Table 5), suggesting that this
effect may be mainly in response to tick feeding. This is corroborated
by the absence of enrichment during A. phagocytophilum transmission
versus feeding by uninfected ticks ([176]Table 3). Interestingly, early
experiments on sheep demonstrated that A. phagocytophilum infection led
to higher number of neutrophils in the skin in the presence or absence
of tick feeding [[177]17]; whether A. phagocytophilum alone triggers
neutrophil chemotaxis to the skin remains to be determined. Studies in
the 1970s suggested that neutrophils may be involved in the pathology
of tick bites of ixodid ticks [[178]43]; nevertheless, what function
neutrophils play during A. phagocytophilum transmission remains
unexplored.
Among other immune pathways affected by the transmission of A.
phagocytophilum in the skin was an increased activation of genes
involved in interferon signaling ([179]Table 3 and [180]Table 5 and
[181]Figure 6). Type I (17 members, including Ifn-α and Ifn-β) and type
II (Ifn-γ) interferons are important against viral and bacterial
infections [[182]44,[183]45]. Their interaction with receptors within
the membrane of cells activate signaling cascades that lead to the
expression of immune-related genes [[184]44]. The importance of Ifn-γ
signaling in the immune response against systemic A. phagocytophilum
infection has been demonstrated in several studies
[[185]10,[186]11,[187]12,[188]13,[189]14]. However, the significance of
these cytokines and signaling pathways during early infection is
unknown. Type I and type II interferon signaling pathways confer
resistance to R. parkeri infection in murine models [[190]46]. Double
knockout mice with mutations in the type I IFN receptor (Ifnar1 −/−)
and Ifn-γ receptor (Ifngr1 −/−) resulted in eschar development in the
skin and lethality after intradermal (i.d.) infection of as low as 10^2
bacteria when compared with 10^7 when injected intravenously (i.v.),
suggesting that IFN signaling in the skin may be important for
protective immunity [[191]46,[192]47]. Furthermore, infection and
vaccination with mutant R. parkeri strains showed that Ifnar1 −/−;
Ifngr1 −/− double knockout mice may be a good tool for the study of
rickettsial pathogenesis, and as models to evaluate vaccine candidates
[[193]46]. As with SFG Rickettsia, A. phagocytophilum does not produce
disease in mice. Given our results, and that bacterial numbers increase
during systemic infection of A. phagocytophilum in Ifn-γ −/− mice
[[194]11], it is possible that these receptors are also important for
initial immune responses in the skin. However, more studies are needed
to understand the role of IFN signaling during bacterial establishment.
Figure 6.
[195]Figure 6
[196]Open in a new tab
Interferon gamma and alpha/beta signaling related genes upregulated
during Anaplasma phagocytophilum transmission. Components in light blue
represent genes that are upregulated during transmission of A.
phagocytophilum when compared with the bite site from uninfected ticks.
Purple proteins are encoded by genes that were not differentially
expressed. Figure created with BioRender based on pathway enrichment
analysis from Reactome.
Unexpectedly, A. phagocytophilum transmission also resulted in the
downregulation of extracellular matrix (ECM) organization related genes
([197]Table 4). Several genes encoding ECM structural components, such
as collagen, thrombospondin-5, integrin ITGA10, Matrilin 3, aggrecan,
elastin, fibronectin, and fibrillin, were downregulated during A.
phagocytophilum transmission by unknown mechanisms ([198]Figure 5;
[199]Supplementary File S4). Enrichment of genes involved in
collagen-related pathways was also observed when DEGs unique to
Anaplasma phagocytophilum transmission, when compared with intact skin,
were analyzed ([200]Table S7). A liver-derived
protease—plasmin—degrades collagens, fibronectin, and other components
of the ECM. Several pathogens, including Borrelia spp., exploit plasmin
to facilitate their invasion into tissues [[201]48]. Interestingly,
α2-macroglobulin (A2m), a protein involved in the regulation of plasmin
to avoid excessive proteolysis, is downregulated in the skin during A.
phagocytophilum transmission ([202]Supplementary File S4). Whether this
results in more damage in the ECM due to plasmin activity during A.
phagocytophilum early infection remains to be determined. A second
mechanism of ECM degradation that is manipulated by pathogens is the
induction of host metalloproteinases [[203]48]. Anaplasma
phagocytophilum is known to stimulate the release of metalloproteinases
during infection of neutrophils and during coinfection with Borrelia
burgdorferi [[204]49,[205]50]. We detected the upregulation of several
metalloproteinases in the site of A. phagocytophilum transmission,
including MMP1b, MMP8, MMP13, and MMP25 ([206]Supplementary File S1).
Both Granquist et al. [[207]16] and Reppert et al. [[208]17] reported
the presence of infected neutrophils within the bite site of infected
ticks and uninfected ticks on experimentally infected hosts,
respectively. Although it is possible that infected neutrophils may be
the source of this upregulation, the synergistic effect of tick saliva
was also detected as these metalloproteinases were also upregulated in
the tick-only control, except for MMP1b ([209]Supplementary Files S4
and S5). Increased permeability of the ECM may facilitate the
dissemination of the infection, warranting the further exploration of
the molecular mechanisms and effects of the downregulation of ECM
integrity genes.
5. Conclusions
Overall, our results indicate that A. phagocytophilum transmission
leads to the activation of interferon signaling pathways and the
upregulation of Th1 cytokines such as Tnfα, Ifn-γ, and other cytokines
and chemokines associated with inflammation in the skin. The
inflammatory environment developed in the skin, results in the
activation of genes involved in neutrophil chemotaxis, confirming
previously reported findings. Further, transmission of this tick-borne
pathogen also causes a downregulation of genes involved in ECM
integrity and an upregulation of metalloproteinases that may increase
vascular leakage, indicating that A. phagocytophilum transmission and
early infection actively delays wound healing responses and may affect
vascular permeability at the bite site. Understanding the initial
responses at the site of transmission of tick-borne pathogens can
assist us to discover protective signaling pathways and identify models
that can be used to distinguish factors that define disease
pathogenesis and protective immunity, as previously shown with R.
parkeri [[210]46].
Acknowledgments