An essential role for miR-15/16 in Treg suppression and restriction of proliferation.

The miR-15/16 family targets a large network of genes in T cells to restrict their cell cycle, memory formation, and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained response. Here, we used conditional deletion of miR-15/16 in regulatory T cells (Tregs) to identify immune functions of the miR-15/16 family in T cells. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16 deficiency alters expression of critical Treg proteins and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 shifts Treg fate and produces an effector Treg phenotype. These Tregs fail to control immune activation, leading to spontaneous multi-organ inflammation and increased allergic inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance.

IL-31-dependent neurogenic inflammation restrains cutaneous type 2 immune response in allergic dermatitis.

Despite robust literature associating IL-31 with pruritic inflammatory skin diseases, its influence on cutaneous inflammation and the interplay between inflammatory and neurosensory pathways remain unmapped. Here, we examined the consequences of disrupting Il31 and its receptor Il31ra in a mouse model of house dust mite (HDM)-induced allergic dermatitis. Il31-deficient mice displayed a deficit in HDM dermatitis-associated scratching, consistent with its well-established role as a pruritogen. In contrast, Il31 deficiency increased the number and proportion of cutaneous type 2 cytokine-producing CD4+ T cells and serum IgE in response to HDM. Furthermore, Il4ra+ monocytes and macrophages capable of fueling a feedforward type 2 inflammatory loop were selectively enriched in Il31ra-deficient HDM dermatitis skin. Thus, IL-31 is not strictly a proinflammatory cytokine but rather an immunoregulatory factor that limits the magnitude of type 2 inflammatory responses in skin. Our data support a model wherein IL-31 activation of IL31RA+ pruritoceptors triggers release of calcitonin gene-related protein (CGRP), which can mediate neurogenic inflammation, inhibit CD4+ T cell proliferation, and reduce T cell production of the type 2 cytokine IL-13. Together, these results illustrate a previously unrecognized neuroimmune pathway that constrains type 2 tissue inflammation in the setting of chronic cutaneous allergen exposure and may explain paradoxical dermatitis flares in atopic patients treated with anti-IL31RA therapy.

An essential role for miR-15/16 in Treg suppression and restriction of proliferation.

The miR-15/16 family is a highly expressed group of tumor suppressor miRNAs that target a large network of genes in T cells to restrict their cell cycle, memory formation and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained immune response. Here, using conditional deletion of miR-15/16 in immunosuppressive regulatory T cells (Tregs) that express FOXP3, we identify new functions of the miR-15/16 family in T cell immunity. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16-deficiency alters Treg expression of critical functional proteins including FOXP3, IL2Rα/CD25, CTLA4, PD-1 and IL7Rα/CD127, and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 inhibition of cell cycle programs shifts Treg diversity and produces an effector Treg phenotype characterized by low expression of TCF1, CD25 and CD62L, and high expression of CD44. These Tregs fail to control immune activation of CD4+ effector T cells, leading to spontaneous multi-organ inflammation and increased allergic airway inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance.

Innate type 2 immunity controls hair follicle commensalism by Demodex mites.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Bile acid-sensitive tuft cells regulate biliary neutrophil influx.

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney.

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.

Lymph node-resident dendritic cells drive TH2 cell development involving MARCH1.

Type 2 T helper (TH2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in TH2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of TH2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-TH2 effect of MARCH1 relied on lymph node (LN)­resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4+ T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop TH2 cells. These findings suggest that TH2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling.

CISH constrains the tuft-ILC2 circuit to set epithelial and immune tone.

Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue.

Decreased p53 is associated with a decline in asymmetric stem cell self-renewal in aged human epidermis.

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self-renewal in multiple murine tissues. The fate of SC self-renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self-renewal has been observed in inflammatory-mediated hyperproliferation, while increased symmetric SC self-renewal has been observed in cancers. We analyzed SC self-renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age-dependent decrease in epidermal basal layer divisions. The balance of SC self-renewal shifted toward symmetric SC self-renewal, with a decline in asymmetric SC self-renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self-renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin-3 (a p53 activator) returned p53 activity as well as asymmetric SC self-renewal divisions to adult levels. Nutlin-3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin-3 (p53)-induced asymmetric SC self-renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53-induced asymmetric SC divisions.

Targeted Urine Metabolomics for Monitoring Renal Allograft Injury and Immunosuppression in Pediatric Patients.

Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs related to non-immune and immune injuries can cause slow deterioration and premature failure of organ transplants. Diagnosis of these injuries by non-invasive urine monitoring would be a significant clinical advancement for patient management, especially in pediatric cohorts. We investigated the metabolomic profiles of biopsy matched urine samples from 310 unique kidney transplant recipients using gas chromatography-mass spectrometry (GC-MS). Focused metabolite panels were identified that could detect biopsy confirmed acute rejection with 92.9% sensitivity and 96.3% specificity (11 metabolites) and could differentiate BK viral nephritis (BKVN) from acute rejection with 88.9% sensitivity and 94.8% specificity (4 metabolites). Overall, targeted metabolomic analyses of biopsy-matched urine samples enabled the generation of refined metabolite panels that non-invasively detect graft injury phenotypes with high confidence. These urine biomarkers can be rapidly assessed for non-invasive diagnosis of specific transplant injuries, opening the window for precision transplant medicine.