3.Results
3.1. Spontaneous cytokine release signatures in PBMCs reflect
respiratory virome profiles.
Based on the baseline release of 13 out of 22 measured cytokines,
individuals were annotated in principal components (Figure 1 and
Supplementary Data 2). Unsupervised clustering identified two groups
with distinct cytokine release signatures (Figure 1). The first group
(Cluster 1) included individuals (n=37, 72.5%) that were characterized
by low spontaneous cytokine release (Figure 1a-1b), while the second
group (Cluster 2) included individuals (n=14, 27.5%) with high overall
spontaneous cytokine release (Figure 1a-1b). All individual cytokines
contributed to the clustering at a range of 5.5%-7.2% (Supplementary
Figure 1a). The majority (86%) of children in the high cytokine
spontaneous release group (Cluster 2) had prokaryotic dominated viromes
(PVPG)(Specifically, high Shannon diversity and richness of prokaryotic
viruses), in contrast to the low cytokine spontaneous release group
(cluster 1) (Figure 1c) that included evenly distributed children with
all three types of virome (PVPG, EVPG, and AVPG) (p:0.018, Chi-Square
test). These findings were independent of the presence of allergy or
asthma (Supplementary figure 1b-1c). There were no significant
associations between baseline cytokine clusters and the presence of
specific viral families (Picornaviridae, Anelloviridae, and
Siphoviridae) in the upper respiratory tract virome (Supplementary
Figure 2). To further assess baseline responses, a logistic regression
model considering geography (Greece, Poland and Finland), VPG, sex, age
and presence of Picornaviridae and Anelloviridae was performed
(Supplementary Table 3). This showed that EVPG and AVPG were
statistically significant and negatively associated with PVPG (EVPG
p:0.034, beta: -2.92; AVPG p:0.022, -3.01) in the non-stimulated culture
medium (Supplementary Table 4), confirming that the virome groups
reflected the two types of response.
3.2. PBMC bacterial immune signatures have minimal associations
with respiratory virome characteristics.
Bacterially-stimulated samples were annotated in a coordinate plot based
on their cytokine induction profiles (n=17) (Figure 2a & Supplementary
Data 3). Hierarchical clustering identified three clusters of
antibacterial immune responses (Figure 2a and Supplementary Figure 3a).
Cluster 1 (n=15, 30%) included children with low release of bacterial
DNA-stimulated cytokines, despite high response to LPS, especially for
IL-1b, IL-6, TNF, CCL3 and CCL4 (Figure 2b). In contrast, Cluster 2
(n=19, 37%) displayed high release of TNF, CCL3, CCL4, IL-6, IL-27 and
IL-1b when stimulated by bacterial DNA (Figure 2b). Finally, Cluster 3
(n=17, 33%) displayed heterogeneous and generally low cytokine
responses (Figure 2b). Each cluster was characterised according to its
features: Cluster 3 children were identified as low responders, Cluster
1 as intermediate responders, and Cluster 2 as high responders. We then
explored possible correlations with the presence of specific viral
families (Picornaviridae (n=28, 55%), Anelloviridae (n=34, 66%),
Siphoviridae (n=20, 39%)) and the virome profiles groups (VPGs) (Figure
2c) to each type of responder. Neither the presence of different virus
families, nor the type of virome significantly differ across the three
cytokine clusters, i.e. these were independent from the antibacterial
response (Figure 2c). Moreover, antibacterial responses were not
associated with asthma or allergy outcomes (Supplementary Figure 3b-3c).
Nevertheless, in the regression model, the geographical location (Poland
p:<0.001, beta:10.95; p:<0.001, beta:-10.91; Finland
p:<0.001, beta:-9.1; p:<0.001, beta:-11.37) and the
presence of Siphoviridae (p:<0.001, beta:-8.67;
p:<0.001, beta:13.81) had significant inference in high and
low responders respectively (Cluster 2 and 3) over intermediate
responders (cluster 1) regarding their antibacterial responses
(Supplementary 5), suggesting a gradient of bacteriophages in reverse
correlation with responses to bacterial stimuli (i.e. high bacterial
response corresponding to low levels of bacteriophages, etc).
3.3. PBMC antiviral immune signatures correlate with the
presence of Siphoviridae and Picornaviridae in the upper respiratory
virome.
We identified 4 clusters describing PBMC responses to viral stimuli
(Figure 3a and 3b & Supplementary Data 4). IL-1b, CCL3, CCL4 and TNF in
all virus-like stimulants had a major influence in clustering the
samples, among a total of 23 significant conditions (Supplementary
Figure 4a). Cluster 4 children (n=5, 10%) had a high and homogeneous
response to TLR3 (Poly:IC) and TLR7/8 (R848) stimulated cytokines,
namely IL-1b, IL-23a, IL-27, IL-6, CCL3, CCL4, TNF, IFN-a2, IL-25, TNF,
IL-13, but not to rhinovirus A (RV-A) (Figure 3b). In contrast, the
release of IL-1b, IFN-γ, CXCL10, CCL3, CCL4, TNF and IL-17a in response
to RV-A were high in Cluster 2 children (n=11, 21.5%) (Figure 3b).
Children with the lowest overall cytokine responses were grouped on
Cluster 3 (n=14, n=27.5%), while the largest group of subjects
displayed a heterogenous pattern in their responses and were assigned to
Cluster 1 (n=21, 41%) (Figure 3b). Consequently, each group was
characterized as follows: Cluster 4: overall high responders, Cluster 2:
RV-A responders, Cluster 1: intermediate responses, Cluster 3: low
responders.
High responders had significantly higher presence of Picornaviruses
(p-value: 0.036, 95%CI) in their upper airway, in comparison to low
cytokine responders (Figure 3c). This was also observed regarding
Siphoviridae, however with statistically marginal value (p:0.072)
(Figure 3d). No differences were observed considering the presence of
Anelloviridae. The comparison of the virome profiles confirmed a
significantly biased virome composition between children in the
different cytokine clusters (p-value: 0.0004, 95%CI) (Figure 3c). These
associations were not affected by the presence of asthma or rhinitis
(Supplementary Figure 4b-4c).
In the regression model, geographical location influenced the clustering
(Supplementary Table 6). The results confirmed the difference between
RV-A and intermediate responders regarding to the presence of
Siphoviridae and Picornaviridae (p:0.027, beta:2.83; p:<0.001,
-11.63). Additionally, in low responders (Cluster 3) there were
significantly more prokaryotic than eukaryotic viral group types
(p:<0.001, beta:-29.67) (Supplementary Table 6).
3.4. Presence of viral families in the airway and its
association with antibacterial and antiviral cytokine induction in
PBMCs.
To further describe potential associations between antibacterial and
antiviral PBMC responses with viral presence in the nasopharynx, we
investigated cytokine induction levels in the presence of the
Picornaviridae, Siphoviridae and Anelloviridae viral families (Figure
4). Among stimulants, bacterial DNA and LPS were considered for the
antibacterial responses and Poly:IC, R848 and RV-A for the antiviral.
When Picornaviridae were present in the nasopharynx, bacterially
stimulated production of IFN-λ-2, CCL5, IL-12b were low (Figure 5). The
presence of Siphoviridae was also related to low antibacterial responses
(Supplementary Figure 5i), however some inflammatory cytokines (IL6,
CXCL8, CCL4, TNF) were upregulated after LPS stimulation (Supplementary
Figure 5ii). Regarding antiviral responses, the presence of
Picornaviridae (Figure 5), as well as Siphoviridae, (Supplementary
Figure 5iii) were associated with low levels of IFN-λ-2 responses;
Siphoviridae were also associated with reduced IL-7, IL-23a, and IL-12b,
but increased IFN-a2. In contrast, presence of Anelloviridae coincided
with increased production of CCL4, IL-6, IL-27 and IL-10 against
bacterial stimuli and TNF and IL-7 against viral stimuli (Supplementary
Figure 6). In all, Siphoviridae displayed the broadest association with
PBMC derived cytokines following either bacterial or viral stimulation
(Figure 4).
4.
Discussion
This is the first study showing
that antiviral immune responses at a systemic level reflect the upper
airway virome composition during free acute infections periods. Although
most attention is currently given to the mechanisms by which the
microbiota and/or their components shape the immune responses [17],
we are also well aware that host immune responses can regulate microbial
expansion and therefore control microbiota [18].
Analysing the immune status in
combination with the microbiome is thought to be necessary for
understanding the mechanisms involved in microbial influence of clinical
outcome [17]. Another relevant finding of the study is the higher
abundance of particular viral families, such as Picornaviridae and
Siphoviridae, in individuals with low innate interferon responses.
When spontaneous cytokine release was evaluated, high producers were
dominated by prokaryotic virome profiles. There are two, non-mutually
exclusive, possible explanations: one, a high cytokine secretion status
may result in reduction of Anellovirus and Picornavirus presence and
diversity, or, high baseline responses might be the result of concurrent
bacterial expansion, which in turn favours the proliferation of
bacteriophages [19]. However, the latter explanation is less likely,
considering that the extent PBMCs get activated by confronting bacteria
during homeostasis, is minor [20].
Regarding antibacterial responses, we observed a differential response
against LPS versus bacterial-DNA. Although both stimuli activate
antibacterial responses in the cell, they initiate TLR signalling from
distinct locations: LPS does not require internalization to activate the
signal cascade, while bacterial DNA does [21]. This distinct immune
activation pattern has been previously reported [22].
In line with the established understanding of rhinovirus (RV) biology,
we have observed a correlation between the presence of picornaviruses
(mostly RVs) and low levels of IFN-λ2, IL12 and RANTES (CCL5), following
innate immune stimulation [23]. Several studies have suggested that
interferon deficiency is a key mechanism supporting RV replication
[24,25,26] and induction of exacerbations in patients with asthma
[27]. Interestingly, although IFN-λ2 showed decreased levels in
presence of picornaviruses, IFN-α2 protein levels remained unchanged.
IFN-λ2 (IL-28A) belongs to IFN III-type and INF-α2 to IFN I-type. In
contrast to type I IFNs, type III IFNs are not ubiquitously expressed
and are mainly found at barrier epithelial surfaces such as the
respiratory tract where they exhibit unique not-redundant antiviral
functions [37,38].Interestingly, it has been reported that type III
IFNs suppress Th2 responses in experimental asthma in mice [39],
while respiratory viral pathogens have evolved mechanisms to suppress
IFN-λ function or downregulate signalling, underlying their contribution
to respiratory immunity at mucosal barriers [28,36].
Our findings indicate that this is a wider mechanism that controls the
extent of RV presence in the upper airway mucosa, in which RV is a
frequent, but transient visitor [29]. Picornaviruses were also
present in all samples from subjects with high antiviral responses, but
low responses to RV (cluster 4). This can be due to RV-specific defects,
as the ones that have been described on a genetic basis [30].
Bacteriophages, such as Siphoviridae, are involved in the modulation of
bacterial communities and therefore potentially influencing health
outcomes [13,31]. In our cohort, the presence of Siphoviridae was
extensively negatively correlated with both antibacterial DNA and
antiviral cytokine immune responses, while there was a positive
correlation of inflammation-related cytokines (IL6, IL8, TNF) following
LPS stimulation. It is probable that a robust antimicrobial capacity
limits the potential of bacterial growth, consequently reducing
bacteriophage proliferation [32]. This finding may have important
implications, as it suggests a potential role of bacteriophages as
sensitive sensors of host immunity. Although data are scarce, the effect
of bacteriophages on the immune system appears to be mostly indirect,
through their impact on their target bacteria [13,19,31].
Nevertheless, more complex viral-bacterial interactions may contribute
to these observations [13].
It is more challenging to explain the observed positive associations
between the presence of anelloviruses and mostly inflammatory (IL6,
MIP1b) and regulatory (IL10, IL27) cytokines following bacterial, and to
a less extent also viral stimulation. Anelloviruses are apparently
non-pathogenic viruses that have been associated with conditions of
immune suppression [33] and are considered an integral part of the
respiratory virome, particularly in asthma [5].
Anellovirus-dominated profiles as well as anellovirus presence were
equally distributed among both antibacterial and antiviral immune
response clusters, suggesting that anellovirus presence may be
controlled by mechanisms other than TLR-stimulation. It is possible that
inflammatory instead of antiviral responses may facilitate anellovirus
proliferation.
When studying the microbiome, it is challenging to differentiate the
causal host-microbiome associations from secondary microbial changes. In
many immune-related conditions, abnormal viral-bacterial interactions
can be considered as either a cause or a marker of the disease state
[34, 35]. Our results highlight important correlations between the
respiratory virome and immune signatures, however, we cannot establish
causality. A noteworthy observation is that even though the cohort was
comprised of healthy and atopic asthmatic individuals, disease was not a
modifier of the correlations, suggesting a fundamental mechanism of
immune-microbial interaction; or the result of insufficient statistical
power to identify such patterns.
One limitation of the study is the measurement of one post-induction
time point, so we could only describe cross-sectional associations
instead of a complete response curve, due to practical limitations. The
number of samples was moderate, however, we used robust and
state-of-the-art methodology for cytokine measurements as well as for
the characterization of the virome. Subjects from a wide geographic
representation were included. All participating centres followed a
validated and synchronized approach for PBMC cultures with common
training and reagents. The processing and assessment for nasopharyngeal
samples and immune responses was independent.
In conclusion, there are tight parallels between the upper airway virome
and the host immune status and potential innate immune responses. Viral
stimulation has the capacity of directing immune responses, while immune
responses themselves may control microbial composition. The unravelling
of such interactions offers new opportunities for intervention towards
disease prevention.