Late inflammatory monocytes define circulatory immune
dysregulation observed in skin microbiome-stratified atopic dermatitis
To the Editor,
Atopic dermatitis (AD) is a skin inflammatory disorder well described
for significant disease heterogeneity. (1) Previously, we defined
steady-state microbial configurations dermotypes A and B that robustly
reflected heterogeneity in AD clinical severity, cutaneous barrier
properties and skin microbiome composition. (2) Here, we explored
circulating immune dysregulation underlying dermotype-stratified AD.
We performed single-cell RNA sequencing (scRNA-seq) of PBMCs from
healthy subjects with dermotype A (n = 4) and AD patients with
dermotypes A (n = 4) and B (n = 6). UMAP reduction distinguished 13
major immune clusters using transcriptional profile differences,
identified using DISCO CELLiD (3) and top differentially expressed genes
(DEGs; Supplementary Figure 1A ). Major clusters corroborated
well across all groups as a testimony to limited sample distortion by
inter-individual or batch effects (Supplementary Figure 1B ).
Critically, AD patients harboured a marked enrichment in monocyte
population cluster 1, whereby dermotype stratification saw a further
enrichment in dermotype B (Supplementary Figure 1B ).
To reveal finer expression details, we sub-clustered myeloid cells,
obtaining 11 sub-populations whose identities were similarly assigned
following DISCO CELLiD and top DEGs (Supplementary Figure 1C ).
Clusters with low cell counts and not of myeloid origin were removed
prior to down sampling for normalisation, yielding 7 myeloid clusters of
interest. Majority of clusters represent CD14+ monocytes except for
clusters 5 and 7 corresponding to CD16+ monocytes and conventional
dendritic cells (cDC) respectively (Figure 1A ). CD14+ monocytes
segregated into 4 transcriptionally distinct states (Figure 1A
and B ): early activation monocytes expressing proinflammatory alarmins
(S100A8 and S100A12; clusters 0 and 1), transitional
monocytes concomitantly expressing alarmins and chemokines
(S100A12 and CCL3; cluster 2), intermediate monocytes
expressing antigen presentation genes (HLA-DPB1 andHLA-DPA1 ; cluster 3), and late inflammatory monocytes expressing
proinflammatory cytokines (IL1B, CCL3 and TNF ; cluster 4,Supplementary Figure 1D ) (4, 5). Late inflammatory monocytes
(cluster 4) were notably enriched in AD dermotype B (Figure 1A
and C ) and exhibited upregulation in MHC class II (HLA-DQA2,
HLA-DQB1 ), monocyte-related (ITGAM, FGD2 ) and proinflammatory
genes (CCL4L2, CCL3L1 and CCL20 ), suggestive of
inflammatory dysregulation (Figure 1D and Supplementary Table
1 ). Taken together, dermotype stratification addressed part of the
cellular heterogeneity observed in circulating immune profiles,
characterised particularly by late inflammatory monocytes enrichment in
dermotype B.
Complimenting scRNA-seq findings with CytoFLEX (Supplementary
Figure 2A ), unstimulated PBMCs were stained and secretion of
inflammatory cytokines CCL3, CCL4, TNF-α, IL-1β, and IL-8 was monitored
(Supplementary Table 2 ). Cytokine secretion by classical (CD14+
CD16-) monocytes were significantly elevated in AD compared to healthy
controls (Figures 2A, B and Supplementary Figure 2B ).
Atopy-relevant cell types were examined, revealing similar levels
between AD dermotypes A and B but elevated presence of Th2 and Th2a in
AD dermotype B as opposed to healthy controls (Figures 2C and
D ). The reverse was observed for NK and ILC3s, where AD subjects with
dermotype B registered cell deficiencies (Figures 2E and F ).
Comparing between AD dermotypes, IL3Cs were significantly diminished in
dermotype B (Figure 2F ). Healthy and AD subjects harbouring
dermotype A shared similar expression of aforementioned cells,
suggesting that disease alone could not differentiate immune
dysregulation observed. Next, we quantified 92 inflammatory biomarkers
in plasma using Olink Proteomics Proximity Extension Assay (PEA) Target
96 Inflammation Panel. AD subjects recorded significantly higher
expression of TNF-associated cytokines TRANCE and TRAIL, monocyte
chemoattractant MCP-4, IL-18, and IL-2 than healthy controls
(Figures 2G-J and Supplementary Figure 2C ). Noticeably, higher
basal abundance of TRANCE and MCP-4 was observed in AD dermotype B than
dermotype A (Figures 2G and I ), lending additional support to
circulatory immune dysregulation previously observed in inflammatory
monocytes.
Our findings provide unique insights into immune heterogeneity
underlying AD patients stratified by defined skin microbiome
configurations. Definingly, dermotype B is characterised by microbial
dysbiosis with reduced species richness, fewer commensal species and
increased presence of S. aureus virulence genes. (2) Rather than
atopy-relevant Type 2 cells, late inflammatory monocytes demonstrated a
striking enrichment in AD dermotype B, particularly in the upregulation
of proinflammatory cytokine transcripts. Coupled with skin microbial
dysbiosis that increases cutaneous exposure to potent allergic
inflammation inducers such as δ-toxin (2, 6), immune dysregulation
directed by monocytes could predispose AD skin towards flare
predisposition and clinical severity as observed in dermotype B. Basal
secretion levels of proinflammatory cytokines by classical monocytes are
elevated in AD regardless of dermotype, suggesting higher baseline
inflammation driven by disease. While no stimulation was conducted here,
earlier reports have demonstrated that LPS activation of monocytes
upregulates transcriptional expression of proinflammatory cytokines
IL-1β, CCL3 and IL-8. (4) Similarly, heightened proinflammatory
cytokines and monocyte-related proteins were detected in plasma of AD
dermotype B compared to dermotype A subjects, validating scRNA-seq
findings of monocyte-driven immune dysregulation and inflammation.
Collectively, circulatory immune profiling of dermotypes, defined by
alterations in skin microbiota compositions, suggest a
pathophysiological role for inflammatory monocytes. Future studies are
needed to ascertain the functional and mechanistic roles of late
inflammatory monocytes in dermotype-stratified AD. Importantly, our
study highlights the potential clinical utility of dermotype
stratification to indicate the cellular basis for increased inflammation
and exacerbated immune dysregulation, setting precedence for better
understanding of disease prognosis.