Cutaneous psoriasis improves with targeted pathway inhibition, but the homeostatic mechanisms that normally restrain chronic tissue inflammation remain incompletely understood. We single-cell profiled human psoriatic and normal skin resident memory T cell transcriptomes to reveal a gradated transcriptional program of coordinately regulated inflammation-suppressive genes. This program, which is sharply suppressed in psoriatic lesional skin, strikingly restricts Th17 cytokine and other inflammatory mediators on the single-cell level. We have recently shown CRISPR-based deactivation of core components of this inflammation-suppressive program replicates the elevated IL17F, IL26, and IFNG in psoriatic memory T cells deficient in these transcripts, functionally validating their influence. Combinatoric expression analysis establishes a dominant single-cell trajectory of increasingly inflamed psoriatic resident memory T cells but can also distinguish the influence of individual suppressive program transcripts on specific inflammatory mediators. Finally, we find that therapeutic IL23 blockade reduces Th17 cell frequency in psoriatic skin but fails to re-establish expression of this inflammation-suppressive transcriptional program, illustrating how treated lesions may be primed for recurrence upon withdrawal of treatment.

Human inflammation activates dynamically, but the mechanisms underlying such sensitive kinetics remain incompletely understood. We single-cell profiled transcriptomes and epitopes from 24 diverse rashes in adults, including psoriasis, atopic dermatitis, lichen planus, and bullous pemphigoid. The tristetraprolin family members ZFP36 and ZFP36L2 were identified as recurrently, highly repressed transcripts in T cells (logeFC = -0.47 and -0.85 respectively; adjPvals < 0.001). CRISPR-mediated knockdown of these genes induced a greater than 2-fold secretion of multiple inflammatory cytokines in T cells, including TNF-α (p = 0.008), GM-CSF (p = 0.002), IFNγ (p = 0.030), and IL13 (p = 0.044). Interestingly, cytokine induction was greatest preceding stimulation, suggesting tristetraprolins amplify signaling at the onset of inflammation. Supporting this temporal model, we also found that attenuation of ZFP36L2 mRNA precedes induction of cytokine transcripts in stimulated T cells. Collectively, our data implicate tristetraprolins as early, central regulators of inflammation in human skin and suggest an actionable point for therapeutic modulation.

The ZFP36 family (ZFP36s) of RNA-binding proteins (ZFP36 (TTP), ZFP36L1 (L1), ZFP36L2 (L2)) regulates gene expression. Evidence in embryonic fibroblasts suggests that ZFP36s modulates expression of LIF, a putative endometrial regulator critical for embryo implantation. We previously reported a complex relationship between the expression of ZFP36s and LIF in whole human endometrium. The purpose of this study was to examine expression in separated human endometrial epithelium and stroma. Cohort study. Normal subjects (n=36) were randomized to endometrial sampling during proliferative (P), early (ES), mid (MS), or late secretory (LS) phase by urinary LH monitoring. Stroma and epithelium were separated by enzymatic digestion and unigravity sedimentation. Relative expression levels were assessed with quantitative, real-time RT-PCR. Kruskal-Wallis and Spearman correlation were used to compare expression levels and assess relationships between the ZPF36s and LIF. TTP, L1 and L2 were all significantly higher in P and MS phases in epithelium, while stroma demonstrated maximum expression of the ZFP36s during P and LS phases. LIF expression was also up-regulated during LS phase in stroma. Across the cycle, LIF was correlated with all ZFP36s in the stroma-- TTP (r=.65, P=0.0001), L1 (r=.6, p=0.0003) and L2 (r=.5, p=0.004)—while epithelium demonstrated correlation with L2 alone (r=.52, p=0.001). ZFP36s expression is cycle regulated in both epithelium and stroma; however, up-regulation of ZFP36s during the secretory phase occurs first in the epithelium. LIFs differential co-variation with the ZFP36s in epithelium and stroma suggests differential mechanisms of regulation. These data, in the context of previous findings suggests that the regulation (and role) of ZFP36s and LIF may depend upon their spatial and temporal expression within the endometrium.

Prior studies have demonstrated comparable pregnancy outcomes in donor-egg IVF cycles using vitrified oocytes. Our aim was to investigate the laboratory and clinical outcomes using vitrified and then immediately thawed oocytes obtained from women in a range of age-groups undergoing IVF. Prospective, randomized, clinical trial. Non-donor women undergoing IVF were enrolled in the study. Their oocytes were randomly assigned to either routine treatment or freezing with thaw 30-60 min later, and then all oocytes underwent ICSI fertilization at the same time. Only embryos from the freeze/thaw group were transferred. Laboratory outcomes (fertilization rates (FR), embryo quality) were compared among sibling oocytes. Of the 8 enrolled women, the median age was 33 years. The median number of oocytes was 16 (range 12-33) and median maturation rate was 74%. The median thaw survival rate in the vitrified group was 80%. The FR and number of good quality day 3 embryos (>6 cells, no fragmentation) were not different between the fresh and vitrified groups. There were 4 clinical pregnancies (3 on-going and 1 missed abortion).Tabled 1Patients' clinical characteristics and laboratory resultsMedian (minimum, maximum) (n=8)Age (years)33 (27, 36)BMI (Kg/m2)25 (20, 32)AMH (ng/ml)3.6 (1.1, 9.8)Peak E2 level (pg/ml)3,328 (1,663, 4,989)Total # of oocytes16 (12, 33)No. of mature oocytes13 (8, 20)Mturation rate (%)74 (57, 86)Fresh groupVitrified groupP-value†Analyzed by signed rank test.# of oocytes7 (4, 10)7 (4, 10)# of survuved oocytes after thawingN/A5 (2, 7)Survival rate (%)N/A80 (50, 100)Fertilization rate (%)85 (40, 90)75 (50, 100)1.00# of good quality embryos1 (0, 2)1 (0, 2)0.13† Analyzed by signed rank test. Open table in a new tab In non-donor IVF cycles, vitrified oocytes have comparable laboratory and pregnancy outcomes compared to fresh oocytes. Oocyte vitrification appears to be a promising modality for women of all ages, not only donor-cycles.

The ZFP36 family of RNA-binding proteins (tristetraprolin (TTP), ZFP36L1 and ZFP36L2) regulate gene expression. Evidence suggests that this protein family modulates expression of LIF, a putative regulator of embryo implantation. The purpose of our study was to determine the relationships between the expression of ZFP36s and LIF in human endometrium in fertile and infertile women. Prospective case-control study. Normal (NL, n=39) and unexplained infertility (UI, n=39) subjects were randomized to endometrial sampling during proliferative (P), early (ES), mid (MS), or late secretory phases, defined by urine LH detection. Relative LIF and ZFP36s (TTP, ZFP36L1, ZFP36L2) mRNA expression was assessed with quantitative, real-time RT-PCR. Spearman correlation and linear regression were used to assess relationships between LIF and ZPF36s. Wilcoxon rank-sum and Kruskal-Wallis tests were used to compare expression levels. All ZFP36s and LIF levels were significantly higher in MS versus other phases in NL subjects, but did not differ in UI subjects. NL and UI MS LIF expression was similar (9.88[1.27-47.32], 17.93[2.17-26.05], P=1.0). NL subjects showed increased expression from ES to MS (0.27[0.07-1.54], 9.88[1.27-47.32], P=0.004), while UI did not (16.49[0.34-67.79], 17.93[2.17-26.05], P=0.94). All ZFP36s expression increased from ES to MS in NL but remained constant in UI subjects. Each ZFP36s correlated with LIF among NL subjects: TTP (P<0.0001, R2=0.40, slope 0.89); ZFP36L1 (P<0.0001, R2=0.37, slope 20.6); ZFP36L2 (P<0.0001, R2=0.39, slope 7.3). There was no significant co-variation between LIF expression and any of the ZFP36s in UI subjects. Expression of each ZFP36s was cycle regulated in normal subjects and was directly correlated with LIF. This regulation and correlation were both disrupted in infertility. These data, in the context of previous findings suggest a role for ZFP36s in the regulation of endometrial LIF expression and fertility.

OBJECTIVE: In ruminant species, a unique type I interferon (IFN), Interferon-τ, is exclusively secreted by the blastocyst and is required for successful pregnancy. Although there is no pregnancy-specific IFN in humans, endometrial IFN expression may be stimulated by Toll-like receptor 3 (TLR3) ligation. We have previously demonstrated cyclic expression of human endometrial epithelial TLR3, with highest expression during mid- and late secretory phases, suggesting a possible role of TLR3 in embryo implantation. The aim of this study is to determine the effects of TLR3 ligation on expression of implantation-associated genes. DESIGN: Laboratory study. MATERIALS AND METHODS: RL95-2 endometrial epithelial cell line and primary cultures of mid-secretory endometrial epithelial cells were treated in triplicate with 5μg/ml of TLR3 ligand, polyinosinic-polycytidylic acid (PolyI:C), a negative control (PolydI:dC), or carrier for 2, 8, 16, and 24 hours. Relative changes in mRNA expression were measured using Taqman® real time RT-PCR (ddCT method) normalized to constitutive genes GAPDH and PPIA in RL95-2 and primary cells, respectively. PolyI:C effects in RL95-2 cells are completely dependent on TLR3. RESULTS: The table shows the peak change in mRNA expression for each gene studied. Treatment with negative controls, polydI:dC or carrier had no effects. Gene names follow standard HUGO nomenclature with common abbreviations noted. Although LIF and CD55 were not induced in primary cells, baseline expression of each was very high in control cells. Also, IP-10 (CXCL10) induction has been shown previously in response to polyI:C. CONCLUSIONS: TLR3 ligation, known to mediate inflammatory responses to viral products, may also be important in promoting embryo implantation, via induction of IDO, COX-2, RANTES, and IP-10. Further studies are ongoing to investigate the role of TLR3 in embryo implantation. Additionally, this is the first evidence of IFNε expression by human endometrium.Table 1Changes in Gene Expression with TLR3 LigationPeak Increase in Expression (fold change; hours)GeneFunctionRL95-2 CellsPrimary CellsINDO (IDO)Fetal Immunotolerance78x; 8h9x; 8hPTGS2 (COX2)Implantation defect in KO Mice6x; 8h10x; 2hCCL5 (RANTES)Embryo Invasion1900x; 24h86x; 8hLIFEmbryo Attachment50x; 8hNo ChangeCD55 (DAF)Prevents Complement Attack4x; 2hNo ChangeISG15Induced at Implantation Sites23x; 8h7x; 8hIFNB1 (IFNβ)Unknown3700x; 2h120x; 2hIFNE1 (IFNε)UnknownNo ChangeNo Change Open table in a new tab