The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Single-cell profiling of prurigo nodularis demonstrates immune-stromal crosstalk driving profibrotic responses and reversal with nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. OBJECTIVES: This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level. METHODS: Single-cell RNA-sequencing of skin from 15 healthy donors and nonlesional and lesional skin from 6 patients each with PN and atopic dermatitis, combined with spatial-sequencing using the 10x Visium platform. Integration with bulk RNA-sequencing data from patients treated with nemolizumab. RESULTS: This study demonstrates that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of profibrotic responses. This study also demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is predominantly found in the papillary dermis of PN skin. Activation of fibrotic responses is the main distinguishing feature between PN and atopic dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. CONCLUSIONS: This study provides a high-resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin.

Epigenetic Regulation of TLR4 in Diabetic Macrophages Modulates Immunometabolism and Wound Repair.

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound healing. In diabetes, macrophages display a prolonged inflammatory phenotype preventing tissue repair. TLRs, particularly TLR4, have been shown to regulate myeloid-mediated inflammation in wounds. We examined macrophages isolated from wounds of patients afflicted with diabetes and healthy controls as well as a murine diabetic model demonstrating dynamic expression of TLR4 results in altered metabolic pathways in diabetic macrophages. Further, using a myeloid-specific mixed-lineage leukemia 1 (MLL1) knockout (Mll1f/fLyz2Cre+ ), we determined that MLL1 drives Tlr4 expression in diabetic macrophages by regulating levels of histone H3 lysine 4 trimethylation on the Tlr4 promoter. Mechanistically, MLL1-mediated epigenetic alterations influence diabetic macrophage responsiveness to TLR4 stimulation and inhibit tissue repair. Pharmacological inhibition of the TLR4 pathway using a small molecule inhibitor (TAK-242) as well as genetic depletion of either Tlr4 (Tlr4-/- ) or myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) resulted in improved diabetic wound healing. These results define an important role for MLL1-mediated epigenetic regulation of TLR4 in pathologic diabetic wound repair and suggest a target for therapeutic manipulation.

The alarmin S100A12 causes sterile inflammation of the human chorioamniotic membranes as well as preterm birth and neonatal mortality in mice†.

Sterile inflammation is triggered by danger signals, or alarmins, released upon cellular stress or necrosis. Sterile inflammation occurring in the amniotic cavity (i.e. sterile intra-amniotic inflammation) is frequently observed in women with spontaneous preterm labor resulting in preterm birth, the leading cause of neonatal morbidity and mortality worldwide; this condition is associated with increased amniotic fluid concentrations of alarmins. However, the mechanisms whereby alarmins induce sterile intra-amniotic inflammation are still under investigation. Herein, we investigated the mechanisms whereby the alarmin S100A12 induces inflammation of the human chorioamniotic membranes in vitro and used a mouse model to establish a causal link between this alarmin and adverse perinatal outcomes. We report that S100A12 initiates sterile inflammation in the chorioamniotic membranes by upregulating the expression of inflammatory mediators such as pro-inflammatory cytokines and pattern recognition receptors. Importantly, S100A12 induced the priming and activation of inflammasomes, resulting in caspase-1 cleavage and the subsequent release of mature IL-1ß by the chorioamniotic membranes. This alarmin also caused the activation of the chorioamniotic membranes by promoting MMP-2 activity and collagen degradation. Lastly, the ultrasound-guided intra-amniotic injection of S100A12 at specific concentrations observed in the majority of women with sterile intra-amniotic inflammation induced preterm birth (rates: 17% at 200 ng/sac; 25% at 300 ng/sac; 25% at 400 ng/sac) and neonatal mortality (rates: 22% at 200 ng/sac; 44% at 300 ng/sac; 31% at 400 ng/sac), thus demonstrating a causal link between this alarmin and adverse perinatal outcomes. Collectively, our findings shed light on the inflammatory responses driven by alarmins in the chorioamniotic membranes, providing insight into the immune mechanisms leading to preterm birth in women with sterile intra-amniotic inflammation.

Downregulation of calponin 2 contributes to the quiescence of lung macrophages.

Calponin 2 is an actin cytoskeleton-associated regulatory protein that inhibits the activity of myosin-ATPase and cytoskeleton dynamics. Recent studies have demonstrated that deletion of calponin 2 restricts the proinflammatory activation of macrophages in atherosclerosis and arthritis to attenuate the disease progression in mice. Here we demonstrate that the levels of calponin 2 vary among different macrophage populations, which may reflect their adaptation to specific tissue microenvironment corresponding to specific functional states. Interestingly, lung resident macrophages express significantly lower calponin 2 than peritoneal resident macrophages, which correlates with decreased substrate adhesion and reduced expression of proinflammatory cytokines and a proresolution phenotype. Deletion of calponin 2 in peritoneal macrophages also decreased substrate adhesion and downregulated the expression of proinflammatory cytokines. Providing the first line of defense against microbial invasion while receiving constant exposure to extrinsic antigens, lung macrophages need to maintain a necessary level of activity while limiting exaggerated inflammatory reaction. Therefore, their low level of calponin 2 may reflect an important physiological adaption. Downregulation of calponin 2 in macrophages may be targeted as a cytoskeleton-based novel mechanism, possibly via endoplasmic reticulum stress altering the processing and secretion of cytokines, to regulate immune response and promote quiescence for the treatment of inflammatory diseases.

Deletion of calponin 2 attenuates the development of calcific aortic valve disease in ApoE-/- mice.

Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and lacks non-surgical treatment. The pathogenesis of CAVD involves perturbation of valvular cells by mechanical stimuli, including shear stress, pressure load and leaflet stretch, of which the molecular mechanism requires further elucidation. We recently demonstrated that knockout (KO) of Cnn2 gene that encodes calponin isoform 2, a mechanoregulated cytoskeleton protein, attenuates atherosclerosis in ApoE KO mice. Here we report that Cnn2 KO also decreased calcification of the aortic valve in ApoE KO mice, an established model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively attenuated vascular atherosclerosis that shares many pathogenic processes with CAVD, it did not reduce aortic valve calcification in ApoE KO mice. Indicating a function in the pathogenesis of CAVD, calponin 2 participates in myofibroblast differentiation that is a leading step in the development of CAVD. The aortic valves of ApoE KO mice exhibited increased expression of calponin 2 and smooth muscle actin (SMA), a hallmark of myofibroblasts. The expression of calponin 2 increased during myofibroblast-like differentiation of primary sheep aortic valve interstitial cells and during the osteogenic differentiation of mouse myofibroblasts. Cnn2 KO attenuated TGFß1-induced differentiation of myofibroblasts in culture as shown by the lower expression of SMA and less calcification than that of wild type (WT) cells. These findings present calponin 2 as a novel molecular target for the treatment and prevention of CAVD.

An M1-like Macrophage Polarization in Decidual Tissue during Spontaneous Preterm Labor That Is Attenuated by Rosiglitazone Treatment.

Decidual macrophages are implicated in the local inflammatory response that accompanies spontaneous preterm labor/birth; however, their role is poorly understood. We hypothesized that decidual macrophages undergo a proinflammatory (M1) polarization during spontaneous preterm labor and that PPARγ activation via rosiglitazone (RSG) would attenuate the macrophage-mediated inflammatory response, preventing preterm birth. In this study, we show that: 1) decidual macrophages undergo an M1-like polarization during spontaneous term and preterm labor; 2) anti-inflammatory (M2)-like macrophages are more abundant than M1-like macrophages in decidual tissue; 3) decidual M2-like macrophages are reduced in preterm pregnancies compared with term pregnancies, regardless of the presence of labor; 4) decidual macrophages express high levels of TNF and IL-12 but low levels of peroxisome proliferator-activated receptor γ (PPARγ) during spontaneous preterm labor; 5) decidual macrophages from women who underwent spontaneous preterm labor display plasticity by M1↔M2 polarization in vitro; 6) incubation with RSG reduces the expression of TNF and IL-12 in decidual macrophages from women who underwent spontaneous preterm labor; and 7) treatment with RSG reduces the rate of LPS-induced preterm birth and improves neonatal outcomes by reducing the systemic proinflammatory response and downregulating mRNA and protein expression of NF-κB, TNF, and IL-10 in decidual and myometrial macrophages in C57BL/6J mice. In summary, we demonstrated that decidual M1-like macrophages are associated with spontaneous preterm labor and that PPARγ activation via RSG can attenuate the macrophage-mediated proinflammatory response, preventing preterm birth and improving neonatal outcomes. These findings suggest that the PPARγ pathway is a new molecular target for future preventative strategies for spontaneous preterm labor/birth.

Invariant NKT Cell Activation Induces Late Preterm Birth That Is Attenuated by Rosiglitazone.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide. Although intra-amniotic infection is a recognized cause of spontaneous preterm labor, the noninfection-related etiologies are poorly understood. In this article, we demonstrated that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induced late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer-induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. Peroxisome proliferator-activated receptor (PPAR)γ activation, through rosiglitazone treatment, reduced the rate of α-GalCer-induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation, as shown by the downregulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4(+) T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils, and mature dendritic cells to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also upregulated the expression of inflammatory genes at the maternal-fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with noninfection-related preterm labor/birth. Collectively, these results demonstrate that iNKT cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome.

Preterm labor in the absence of acute histologic chorioamnionitis is characterized by cellular senescence of the chorioamniotic membranes.

BACKGROUND: Decidual senescence has been considered a mechanism of disease for spontaneous preterm labor in the absence of severe acute inflammation. Yet, signs of cellular senescence have also been observed in the chorioamniotic membranes from women who underwent the physiological process of labor at term. OBJECTIVE: We aimed to investigate whether, in the absence of acute histologic chorioamnionitis, the chorioamniotic membranes from women who underwent spontaneous preterm labor or labor at term exhibit signs of cellular senescence. STUDY DESIGN: Chorioamniotic membrane samples were collected from women who underwent spontaneous preterm labor or labor at term. Gestational age-matched nonlabor controls were also included. Senescence-associated genes/proteins were determined using reverse transcription quantitative polymerase chain reaction analysis (n = 7-9 each for array; n = 26-28 each for validation), enzyme-linked immunosorbent assays (n = 7-9 each), immunoblotting (n = 6-7 each), and immunohistochemistry (n = 7-8 each). Senescence-associated ß-galactosidase activity (n = 7-11 each) and telomere length (n = 15-22 each) were also evaluated. RESULTS: In the chorioamniotic membranes without acute histologic chorioamnionitis: (1) the expression profile of senescence-associated genes was different between the labor groups (term in labor and preterm in labor) and the nonlabor groups (term no labor and preterm no labor), yet there were differences between the term in labor and preterm in labor groups; (2) most of the differentially expressed genes among the groups were closely related to the tumor suppressor protein (TP53) pathway; (3) the expression of TP53 was down-regulated in the term in labor and preterm in labor groups compared to their nonlabor counterparts; (4) the expression of CDKN1A (gene coding for p21) was up-regulated in the term in labor and preterm in labor groups compared to their nonlabor counterparts; (5) the expression of the cyclin kinase CDK2 and cyclins CCNA2, CCNB1, and CCNE1 was down-regulated in the preterm in labor group compared to the preterm no labor group; (6) the concentration of TP53 was lower in the preterm in labor group than in the preterm no labor and term in labor groups; (7) the senescence-associated ß-galactosidase activity was greater in the preterm in labor group than in the preterm no labor and term in labor groups; (8) the concentration of phospho-S6 ribosomal protein was reduced in the term in labor group compared to its nonlabor counterpart, but no differences were observed between the preterm in labor and preterm no labor groups; and (9) no significant differences were observed in relative telomere length among the study groups (term no labor, term in labor, preterm no labor, and preterm in labor). CONCLUSION: In the absence of acute histologic chorioamnionitis, signs of cellular senescence are present in the chorioamniotic membranes from women who underwent spontaneous preterm labor compared to those who delivered preterm in the absence of labor. However, the chorioamniotic membranes from women who underwent spontaneous labor at term did not show consistent signs of cellular senescence in the absence of histologic chorioamnionitis. These results suggest that different pathways are implicated in the pathological and physiological processes of labor.

HMGB1 Induces an Inflammatory Response in the Chorioamniotic Membranes That Is Partially Mediated by the Inflammasome.

Spontaneous preterm labor occurs in two subsets of patients with sterile intra-amniotic inflammation, a process induced by alarmins such as high-mobility group box-1 (HMGB1). Inflammasomes are implicated in the process of spontaneous preterm labor. Therefore, we investigated whether HMGB1 initiates an inflammasome-associated inflammatory response in the chorioamniotic membranes. Incubation of the chorioamniotic membranes with HMGB1 1) induced the release of mature IL-1beta and IL-6; 2) upregulated the mRNA expression of the pro-inflammatory mediators NFKB1, IL6, TNF, IL1A, IFNG, and HMGB1 receptors RAGE and TLR2; 3) upregulated the mRNA expression of the inflammasome components NLRP3 and AIM2 as well as NOD proteins (NOD1 and NOD2); 4) increased the protein concentrations of NLRP3 and NOD2; 5) increased the concentration of caspase-1 and the quantity of its active form (p20); and 6) upregulated the mRNA expression and active form of MMP-9. In addition, HMGB1 concentrations in chorioamniotic membrane extracts from women who underwent spontaneous preterm labor were greater than in those from women who had undergone spontaneous labor at term. Collectively, these results show that HMGB1 can induce an inflammatory response in the chorioamniotic membranes, which is partially mediated by the inflammasome. These results provide insight into the mechanisms whereby HMGB1 induces preterm labor and birth in mice and explain why the concentration of this alarmin is increased in women who undergo spontaneous preterm labor.

Vaginal progesterone, but not 17α-hydroxyprogesterone caproate, has antiinflammatory effects at the murine maternal-fetal interface.

OBJECTIVE: Progestogen (vaginal progesterone or 17-alpha-hydroxyprogesterone caproate [17OHP-C]) administration to patients at risk for preterm delivery is widely used for the prevention of preterm birth (PTB). The mechanisms by which these agents prevent PTB are poorly understood. Progestogens have immunomodulatory functions; therefore, we investigated the local effects of vaginal progesterone and 17OHP-C on adaptive and innate immune cells implicated in the process of parturition. STUDY DESIGN: Pregnant C57BL/6 mice received vaginal progesterone (1 mg per 200 µL, n = 10) or Replens (control, 200 µL, n = 10) from 13 to 17 days postcoitum (dpc) or were subcutaneously injected with 17OHP-C (2 mg per 100 µL, n = 10) or castor oil (control, 100 µL, n = 10) on 13, 15, and 17 dpc. Decidual and myometrial leukocytes were isolated prior to term delivery (18.5 dpc) for immunophenotyping by flow cytometry. Cervical tissue samples were collected to determine matrix metalloproteinase (MMP)-9 activity by in situ zymography and visualization of collagen content by Masson's trichrome staining. Plasma concentrations of progesterone, estradiol, and cytokines (interferon [IFN]γ, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, keratinocyte-activated chemokine/growth-related oncogene, and tumor necrosis factor-α) were quantified by enzyme-linked immunosorbent assays. Pregnant mice pretreated with vaginal progesterone or Replens were injected with 10 µg of an endotoxin on 16.5 dpc (n = 10 each) and monitored via infrared camera until delivery to determine the effect of vaginal progesterone on the rate of PTB. RESULTS: The following results were found: (1) vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ regulatory T cells; (2) vaginal progesterone, but not 17OHP-C, decreased the proportion of decidual CD8+CD25+Foxp3+ T cells and macrophages; (3) vaginal progesterone did not result in M1→M2 macrophage polarization but reduced the proportion of myometrial IFNγ+ neutrophils and cervical active MMP-9-positive neutrophils and monocytes; (4) 17OHP-C did not reduce the proportion of myometrial IFNγ+ neutrophils; however, it increased the abundance of cervical active MMP-9-positive neutrophils and monocytes; (5) vaginal progesterone immune effects were associated with reduced systemic concentrations of IL-1ß but not with alterations in progesterone or estradiol concentrations; and (6) vaginal progesterone pretreatment protected against endotoxin-induced PTB (effect size 50%, P = 0.011). CONCLUSION: Vaginal progesterone, but not 17OHP-C, has local antiinflammatory effects at the maternal-fetal interface and the cervix and protects against endotoxin-induced PTB.

HERC6 regulates STING activity in a sex-biased manner through modulation of LATS2/VGLL3 Hippo signaling.

Interferon (IFN) activity exhibits a gender bias in human skin, skewed toward females. We show that HERC6, an IFN-induced E3 ubiquitin ligase, is induced in human keratinocytes through the epidermal type I IFN; IFN-κ. HERC6 knockdown in human keratinocytes results in enhanced induction of interferon-stimulated genes (ISGs) upon treatment with a double-stranded (ds) DNA STING activator cGAMP but not in response to the RNA-sensing TLR3 agonist. Keratinocytes lacking HERC6 exhibit sustained STING-TBK1 signaling following cGAMP stimulation through modulation of LATS2 and TBK1 activity, unmasking more robust ISG responses in female keratinocytes. This enhanced female-biased immune response with loss of HERC6 depends on VGLL3, a regulator of type I IFN signature. These data identify HERC6 as a previously unrecognized negative regulator of ISG expression specific to dsDNA sensing and establish it as a regulator of female-biased immune responses through modulation of STING signaling.

Oral Baricitinib in the Treatment of Cutaneous Lichen Planus.

Background: Cutaneous lichen planus (LP) is a recalcitrant, difficult-to-treat, inflammatory skin disease characterized by pruritic, flat-topped, violaceous papules on the skin. Baricitinib is an oral Janus kinase (JAK) 1/2 inhibitor that interrupts the signaling pathway of interferon (IFN)-γ, a cytokine implicated in the pathogenesis of LP. Methods: In this phase II trial, twelve patients with cutaneous LP received baricitinib 2 mg daily for 16 weeks, accompanied by in-depth spatial, single-cell, and bulk transcriptomic profiling of pre-and post-treatment samples. Results: An early and sustained clinical response was seen with 83.3% of patients responsive at week 16. Our molecular data identified a unique, oligoclonal IFN-γ, CD8+, CXCL13+ cytotoxic T-cell population in LP skin and demonstrate a rapid decrease in interferon signature within 2 weeks of treatment, most prominent in the basal layer of the epidermis. Conclusion: This study demonstrates the efficacy and molecular mechanisms of JAK inhibition in LP. Trial Registration Number : NCT05188521.

Systems-based identification of the Hippo pathway for promoting fibrotic mesenchymal differentiation in systemic sclerosis.

Systemic sclerosis (SSc) is a devastating autoimmune disease characterized by excessive production and accumulation of extracellular matrix, leading to fibrosis of skin and other internal organs. However, the main cellular participants in SSc skin fibrosis remain incompletely understood. Here using differentiation trajectories at a single cell level, we demonstrate a dual source of extracellular matrix deposition in SSc skin from both myofibroblasts and endothelial-to-mesenchymal-transitioning cells (EndoMT). We further define a central role of Hippo pathway effectors in differentiation and homeostasis of myofibroblast and EndoMT, respectively, and show that myofibroblasts and EndoMTs function as central communication hubs that drive key pro-fibrotic signaling pathways in SSc. Together, our data help characterize myofibroblast differentiation and EndoMT phenotypes in SSc skin, and hint that modulation of the Hippo pathway may contribute in reversing the pro-fibrotic phenotypes in myofibroblasts and EndoMTs.

Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling.

IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator, and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α stimulation) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner, thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator, also increased following TCF4 siRNA knockdown, and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together, these findings identify a role for TCF4 in the negative regulation of IL-17C, which, alone and with TNF-α and IL-17A, feed back to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.

Single-cell sequencing reveals Hippo signaling as a driver of fibrosis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players and defining their interactions. We found a striking layering of the chronic HS infiltrate and identified the contribution of 2 fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response. We further demonstrated the central role of the Hippo pathway in promoting extensive fibrosis in HS and provided preclinical evidence that the profibrotic fibroblast response in HS can be modulated through inhibition of this pathway. These data provide insights into key aspects of HS pathogenesis with broad therapeutic implications.

Targeting immune cell trafficking and vascular endothelial cells in psoriasis.

The role of the vasculature in inflammatory skin disorders is an exciting area of investigation. Vascular endothelial cells (ECs) play instrumental roles in maintaining the vascular barrier and control of blood flow. Furthermore, ECs contribute to a variety of immune responses, such as targeting immune cells to specific areas of vascular damage, infection, or foreign material. However, mechanisms through which ECs participate in immune-mediated responses remain to be fully explored. In this issue of the JCI, Li, Shao, et al. report on vascular endothelial glycocalyx destruction and the mechanisms through which EC dysfunction contributes to the well-characterized immune-mediated features of psoriasis, a chronic inflammatory skin disease. Here, we discuss the implications of these findings and highlight some risks and benefits of existing therapies designed to target immune cell trafficking in a variety of inflammatory conditions.

Placental transcriptome analysis of hypertensive pregnancies identifies distinct gene expression profiles of preeclampsia superimposed on chronic hypertension.

BACKGROUND: The pathogenesis of preeclampsia superimposed on chronic hypertension (SI) is poorly understood relative to preeclampsia (PreE) occurring in pregnant people without chronic hypertension. Placental transcriptomes in pregnancies complicated by PreE and SI have not been previously compared. METHODS: We identified pregnant people in the University of Michigan Biorepository for Understanding Maternal and Pediatric Health with hypertensive disorders affecting singleton, euploid gestations (N = 36) along with non-hypertensive control subjects (N = 12). Subjects were grouped as: (1) normotensive (N = 12), (2) chronic hypertensive (N = 13), (3) preterm PreE with severe features (N = 5), (4) term PreE with severe features (N = 11), (5) preterm SI (N = 3), or (6) term SI (N = 4). Bulk RNA sequencing of paraffin-embedded placental tissue was performed. The primary analysis assessed differential gene expression relative to normotensive and chronic hypertensive placentas, where Wald adjusted P values < 0.05 were considered significant. Unsupervised clustering analyses and correlation analyses were performed between conditions of interest, and a gene ontology was constructed. RESULTS: Comparing samples from pregnant people with hypertensive diseases to non-hypertensive controls, there were 2290 differentially expressed genes. The log2-fold changes in genes differentially expressed in chronic hypertension correlated better with term (R = 0.59) and preterm (R = 0.63) PreE with severe features than with term (R = 0.21) and preterm (R = 0.22) SI. A relatively poor correlation was observed between preterm SI and preterm PreE with severe features (0.20) as well as term SI and term PreE with severe features (0.31). The majority of significant genes were downregulated in term and preterm SI versus normotensive controls (92.1%, N = 128). Conversely, most term and preterm PreE with severe features genes were upregulated compared to the normotensive group (91.8%, N = 97). Many of the upregulated genes in PreE with the lowest adjusted P values are known markers of abnormal placentation (e.g., PAAPA, KISS1, CLIC3), while the downregulated genes with the greatest adjusted P values in SI have fewer known pregnancy-specific functions. CONCLUSIONS: We identified unique placental transcriptional profiles in clinically relevant subgroups of individuals with hypertension in pregnancy. Preeclampsia superimposed on chronic hypertension was molecularly distinct from preeclampsia in individuals without chronic hypertension, and chronic hypertension without preeclampsia, suggesting that preeclampsia superimposed on hypertension may represent a distinct entity.

Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.

CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.

Pansclerotic morphea is characterized by IFN-γ responses priming dendritic cell fibroblast crosstalk to promote fibrosis.

Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and to identify therapeutically targetable pathways by performing single-cell and spatial RNA-Seq on 7 healthy controls and on lesional and nonlesional skin biopsies of a patient with PSM 12 months apart. We then validated our findings using immunostaining and in vitro approaches. Fibrotic skin was characterized by prominent type II IFN response, accompanied by infiltrating myeloid cells, B cells, and T cells, which were the main IFN-γ source. We identified unique CXCL9+ fibroblasts enriched in PSM, characterized by increased chemokine expression, including CXCL9, CXCL10, and CCL2. CXCL9+ fibroblasts were related to profibrotic COL8A1+ myofibroblasts, which had enriched TGF-ß response. In vitro, TGF-ß and IFN-γ synergistically increased CXCL9 and CXCL10 expression, contributing to the perpetuation of IFN-γ responses. Furthermore, cell-to-cell interaction analyses revealed cDC2B DCs as a key communication hub between CXCL9+ fibroblasts and COL8A1+ myofibroblasts. These results define PSM as an inflammation-driven condition centered on type II IFN responses. This work identified key pathogenic circuits between T cells, cDC2Bs, and myofibroblasts, and it suggests that JAK1/2 inhibition is a potential therapeutic option in PSM.