Activation of STAT signaling in SLE patients
Peripheral anticoagulated blood cells were lysed, fixed, and stained with antibodies specific for the surface markers CD3, CD14, and CD19. The phosphorylation of STAT1, STAT3, and STAT5 on the activating residues was measured and compared in three subsets of immune cells (CD3+ T cells, CD19+ B cells, and CD14+ monocytes) in 20 SLE patients, 9 RA patients and 13 HDs (). The SLE patients, RA patients and HDs did not differ significantly with respect to their mean ages or sex distributions (). The mean SLEDAI-2K score of 7.35 indicates that the SLE patients had moderate disease activity (). The median fluorescence intensity (MFI) for each pSTAT in the SLE, RA patients was calculated and compared with that of the HDs.
pSTATs expression in SLE patient and healthy donor (HD).
Increased activation of STAT3 was observed in SLE, RA T cells (SLE 3.584±0.1243, RA 3.620±0.2553, HD 3.034±0.1026, SLE vs HD, P<0.01, RA vs HD P<0.05, , Revised ). SLE monocytes had significantly higher activation of STAT3 than disease control RA and healthy donors. The expression level of phosphorylation of STAT3 in Monocytes of RA showed lower than SLE and HD (SLE 10.25±0.5551, RA 6.946±0.4587, HD 8.648±0.3694, SLE vs HD P<0.05, SLE vs RA, P<0.01, RA vs HD, P<0.01, , Revised ). Increased activation of STAT5 was observed in SLE, RA B cells and T cells. (B cells: SLE 4.837±0.2955, RA 4.945±0.2672, HD 3.866±0.1453, SLE vs HD P<0.05, RA vs HD P<0.01, , Revised 2C; T cells: SLE 5.889±0.2864, RA 4.774±0.4005, HD 3.034±0.1026, SLE vs HD, P<0.001, RA vs HD P<0.01, , Revised ). The expression level of phosphorylation of STAT5 in T cells showed lower in RA than SLE (P<0.05, Revised ). Our data indicated the activation of STAT3, STAT5 signaling in PBMC of SLE patients. There existed disease-specific profiling of PBMC pSTATs. There was activation of STAT3 in the monocytes of SLE patients, but this STAT3 signaling was inhibited in the monocytes of RA patients (Revised ).
Comparison of pSTATs expression between SLE, RA patients and healthy donors (HDs).
No STAT1 activation was detected in any of the three major immune-cell types.
Activation of basal STAT signaling is associated with SLE activity
The basal STAT signaling transduction varied significantly within one SLE patient, and between the SLE patient samples. The activation of STAT3 was increased in SLE T cells and monocytes, as was the activation of STAT5 in SLE T cells and B cells, relative to that in HDs. STAT signaling increased in some patients but decreased or disappeared in others. For example, SLE patient 10 showed STAT5 activation in his B cells and T cells, but lower pSTAT3 was detected in his T cells and monocytes. In SLE patient 01, STAT5 was activated in his T cells, but the phosphorylation of STAT5 was lower in his B cells, and pSTAT3 was reduced in his T cells and monocytes ().
SLE samples were clustered by the expression of basal phosphorylated STAT3 and STAT5.
The SLE patients were further clustered with the HCE3.5 package (Hierarchical Cluster Explorer), according to the basal activation of the STAT3 in their mononuclear cells and T cells, and of STAT5 in their B cells and T cells. Each grid represents the phosphorylation of a STAT protein in one cell type. The phosphorylation of each STAT protein was scaled relative to the minimum phosphorylation level among the 20 SLE samples. Unsupervised clustering identified two main groups of SLE patients (). The patients of group 1 were primarily defined by potentiated STAT5 signaling in their T cells and B cells, whereas there was no detectable activation of STAT5 in the B cells of the patients in group 2.
To investigate whether the phospho-profiling of STAT proteins might be related to lupus activity, we compared the clinical manifestations of the two groups identified with hierarchical clustering (). SLEDAI-2K and the erythrocyte sedimentation rate (ESR) were higher in the patients of group 2 (9.444±1.98 and 45.43±8.96 mm/h, respectively) than those in group 1 (4.364±0.9271 and 16±3.613 mm/h, respectively) (P
0.0237 and P
0.0052, respectively). The patients in group 2 had lower C3 levels than those of group 1, but this difference was not significant.
Comparison of SLE patients in the two clusters.
No significant differences was found in either the clinical manifestations, such as rash, arthritis, vasculitis, oral ulcers, proteinuria, white blood cells, and hemoglobin, or the autoantibody profiles, including anti-Ro, anti-U1 RNP, anti-Sm, and anti-dsDNA, of the two groups. There was no statistically significant difference in the prednisone doses of the two groups. Hydroxychloroquine (HCQ) was used more commonly by group 1 patients than by group 2 patients (P
0.0281; , ).
To determine whether the activation of basal STAT signaling varies over time and parallels SLE activity, the cell-specific phosphorylation of STATs in four SLE patients with active lupus were re-analyzed after clinical remission. There was a positive correlation between STAT5 activity and lupus activity as assessed by SLEDAI-2K ().
Phosphorylation of STAT5 was observed in patients at the beginning and after treatment.
STAT5 signaling in B cells is associated with inflammatory cytokines
STAT5 activation in B cells correlated with the activity of SLE, as estimated as SLEDAI-2K. We then asked whether the inflammatory cytokines influence STAT5 signaling in lupus patients. A panel of 27 cytokines was detected in the sera of SLE patients using Bio-Plex™ Cytokine Assays. A series of cytokines, including IL2 (P
0.44 ; ), IFNγ (P
0.45; ), and G-CSF (P
0.37; ) were all correlated positively with STAT5 phosphorylation in B cells.
Association between the mean fluorescence index (MFI) of pSTAT5 in B cells and cytokine levels in SLE patients.
Phosphorylation of Stat5 in T cells is associated with serum prolactin
PRL–STAT5 signaling was observed in the pathogenesis of cancer, including breast cancer 
. In this study, we found high STAT5 activity in the T cells and B cells of SLE patients. To identify whether serum PRL levels are associated with the activation of STAT5 signaling in T and B cells, we compared the percentage of T cells and B cells with STAT5 activity and serum PRL levels. The percentage of T cells with phosphorylated STAT5 correlated positively with serum PRL (r2
Reduced STAT signaling in response to IFNα and IL6 cytokines
Most cytokines activate STAT proteins that are phosphorylated at certain residues when they combined with specific surface receptors on the immune cells. We found basal levels of STAT3 and STAT5 signaling activation in SLE patients. Quantifying the cell-type-specific STAT signaling responses to cytokines should help us to better understand the pathology of SLE. PBMC were stimulated ex vivo with the inflammatory cytokines IFNα, IFNγ, IL2, IL6, and IL10, which are reported to play a role in the pathogenesis of SLE. Cytokine-induced fold changes in STAT phosphorylation were compared with those of HDs.
In general, there was a reduced signaling response to certain cytokines in SLE patients compared with those in HDs. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the activation of STAT1 in monocytes. The STAT3 response to IL6 was reduced in SLE T cells. Although there was enhanced responsiveness to IL10, but this difference in SLE patients was not statistically significant (, ).
Comparison pSTATs expression between SLE patient and healthy donor (HD) after cytokine stimulation.
pSTATs in SLE patients after cytokine stimulation.