Immunoprofiling of monocytes in STAT1 gain-of-function chronic mucocutaneous candidiasis

Abstract

Patients with STAT1 gain-of-function (GOF) mutations suffer from an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). The pathogenesis behind this complex and heterogeneous disease is still incompletely understood. Beyond the well-recognized Th17 failure, linked to the STAT1/STAT3 dysbalance-driven abrogation of antifungal defense, only little is known about the consequences of augmented STAT1 signaling in other cells, including, interestingly, the innate immune cells. STAT1-mediated signaling was previously shown to be increased in STAT1 GOF CD14+ monocytes. Therefore, we hypothesized that monocytes might represent important co-orchestrators of antifungal defense failure, as well as various immunodysregulatory phenomena seen in patients with STAT1 GOF CMC, including autoimmunity. In this article, we demonstrate that human STAT1 GOF monocytes are characterized by proinflammatory phenotypes and a strong inflammatory skew of their secretory cytokine profile. Moreover, they exhibit diminished CD16 expression, and reduction of classical (CD14++C16-) and expansion of intermediate (CD14++16+) subpopulations. Amongst the functional aberrations, a selectively enhanced responsiveness to TLR7/8 stimulation, but not to other TLR ligands, was noted, which might represent a contributing mechanism in the pathogenesis of STAT1 GOF-associated autoimmunity. Importantly, some of these features extend to STAT1 GOF monocyte-derived dendritic cells and to STAT1 GOF peripheral myeloid dendritic cells, suggesting that the alterations observed in monocytes are, in fact, intrinsic due to STAT1 mutation, and not mere bystanders of chronic inflammatory environment. Lastly, we observe that the proinflammatory bias of STAT1 GOF monocytes may be ameliorated with JAK inhibition. Taken together, we show that monocytes likely play an active role in both the microbial susceptibility and autoimmunity in STAT1 GOF CMC.

Keywords: STAT1; candidiasis; cmc; dendritic cell; immunodeficiencies; monocytes; ruxolitinib.

Figure 1

STAT1 mutations in the patient cohort. (A) STAT1 mutations in the 3D dimeric protein structure; the position of each mutation is highlighted (B) Representative histogram of STAT1 phosphorylation (Tyr701) upon IFNα stimulation in STAT1 GOF and HDs’ monocytesdetected by flow cytometry (C) Kinetics of STAT1 and STAT3 phosphorylation (Tyr701) upon IFNα stimulation in STAT1 GOF (n = 3) and HDs’ monocytes (n = 4) detected by flow cytometry (D) Phosphorylation of STAT1 (pSTAT1; Tyr701) and STAT3 (pSTAT3; Tyr705) upon IFNγ and IFNα stimulation in STAT1 GOF (n = 7) and HDs’ monocytes (n = 11) detected by flow cytometry HD - healthy donors; Tyr - tyrosine. Values are standardized and expressed as median values. Statistical analyses were performed using paired t-tests. Values of p<0.05 (*), p<0.01 were considered statistically significant.

Figure 2

Monocyte characteristics. (A) Radar graph of STAT1 GOF (n = 7) and HDs’ (n = 8) monocyte phenotype and basal cytokine production (B) Cytokine production of STAT1 GOF (n = 7) and HDs’ (n = 8) monocytes detected by flow cytometry (C) Phenotype of STAT1 GOF (n = 7) and HDs’ (n = 8) monocytes detected by flow cytometry (D) Representative dot plot of monocyte subset determination (E) Representative dot plot of monocyte subset determination utilizing CCR2 expression (F) CD16 expression on monocyte subsets surface of STAT1 GOF (n = 7) and HDs (n=8) detected by flow cytometry (G) Distribution of monocyte subsets in STAT1 GOF (n = 7) and HDs (n = 8) (H) Quantification of monocyte subsets in STAT1 GOF (n = 7) and HDs (n = 8) (I) CD86 expression on monocyte surface upon stimulation of various TLR ligands for 24hours in STAT1 GOF (n=7) and HDs (n=9) detected by flow cytometry (J) CXCL10 production by monocytes upon R848 (1ug/ml) stimulation in STAT1 GOF (n = 7) and HDs (n=8) detected by flow cytometry HD - healthy donors. Values are standardized and expressed as median values. Statistical analyses were performed using paired t-tests. Values of p<0.05 (*), p<0.01 (**), and p<0.0001 (****) were considered statistically significant.

Figure 3

Dendritic cells characteristics (A) Gating strategy for DC2 and DC3 subsets analysis (B) Gating strategy forDC1 and DC4 determination (C) Color-coded gating strategy identifying DC subsets in STAT1 GOF patients (n = 5) and HDs (n = 5). The individual subsets are depicted as manually gated populations overlaid onto the tSNE plots (D) Representative tSNE plots of 5 STAT1 GOF and 5 HDs’ DC showing expression of CD14, D16 and CD123. The DC’s subsets are depicted as manually gated populations overlaid onto the tSNE plots (E) pDC and mDC quantification in STAT1 GOF (n = 7) and HDs (n = 9) detected by flow cytometry (F) mDC phenotype of STAT1 GOF (n = 7) and HDs’ DC (n = 9) detected by flow cytometry(G) Quantification of DCs’ subsets in STAT1 GOF and HDs detected by flow cytometry (H) CD86 expression on mDCs’ surface upon stimulation with various TLR ligands for 24hours in STAT1 GOF (n = 7) and HDs (n = 9) detected by flow cytometry (I) CXCL10 production by mDCs upon R848 (1ug/mL) stimulation in STAT1 GOF (n=7) and HD (n=8) detected by flow cytometry DC, dendritic cell; HD, healthy donors; tSNE, t-distributed stochastic neighbor embedding. Values are standardized and expressed as median values. Statistical analyses were performed using paired t-tests. Values of p<0.05 (*), and p<0.0001 (****) were considered statistically significant.

Figure 4

Monocyte-derived derived dendritic cells model (A) Expression of CD86, CD40 and PD-L1 on moDCs’ surface of STAT1 GOF (n = 3) and HDs (n = 3) detected by flow cytometry (B) Cytokine production of STAT1 GOF (n = 3) and HDs’ (n = 3) moDCs detected by LUMINEX (C) CD16 expression on moDCs’ surface of STAT1 GOF (n = 3) and HDs (n = 3) detected by flow cytometry.

Figure 5

Serum cytokines/chemokines/growth factors (A) Heat map showing differences in serum levels of various cytokines, chemokines and GFs between STAT1 GOF patients (n = 5), ruxolitinib-treated STAT1 GOF patients (STAT1 GOF RUXO) (n = 3) and HDs (n = 7) detected by LUMINEX (B) Principal component analysis showing differences in serum levels of various cytokines, chemokines and GFs between STAT1 GOF patients (n=5), ruxolitinib-treated STAT1 GOF patients (STAT1 GOF RUXO) (n = 3) and HDs (n = 7) (C) Dynamics of analytes in a patient before and on ruxolitinib treatment (D) Differences in serum cytokine levels between ruxolitinib treated STAT1 GOF patients (n = 3) and untreated patients(n = 5) (E) Comparison of basal cytokine production by monocytes in HD, STAT1 GOF patient and the ruxolitinib-treated patient (F) Monocyte’s phenotype in HD, STAT1 GOF patient and the ruxolitinib-treated patient detected by flow cytometry HD - healthy donors; GF - growth factor. Values are standardized and expressed as median values. Statistical analyses were performed using paired t-tests. Values of p<0.05 (*), were considered statistically significant.