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.

Transcriptomic characterization of prurigo nodularis and the therapeutic response to nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a debilitating, difficult-to-treat, intensely pruritic, chronic inflammatory skin disease characterized by hyperkeratotic skin nodules. The pathogenesis of PN is not well understood but is believed to involve cross talk between sensory nerve fibers, immune cells, and the epidermis. It is centered around the neuroimmune cytokine IL-31, driving an intractable itch-scratch cycle. OBJECTIVE: We sought to provide a comprehensive view of the transcriptomic changes in PN skin and characterize the mechanism of action of the anti-IL-31 receptor inhibitor nemolizumab. METHOD: RNA sequencing of biopsy samples obtained from a cohort of patients treated with the anti-IL-31 receptor inhibitor nemolizumab and taken at baseline and week 12. Generation and integration of patient data with RNA-Seq data generated from reconstructed human epidermis stimulated with IL-31 and other proinflammatory cytokines. RESULTS: Our results demonstrate that nemolizumab effectively decreases IL-31 responses in PN skin, leading to effective suppression of downstream inflammatory responses including TH2/IL-13 and TH17/IL-17 responses. This is accompanied by decreased keratinocyte proliferation and normalization of epidermal differentiation and function. Furthermore, our results demonstrate how transcriptomic changes associated with nemolizumab treatment correlate with improvement in lesions, pruritus, stabilization of extracellular matrix remodeling, and processes associated with cutaneous nerve function. CONCLUSION: These data demonstrate a broad response to IL-31 receptor inhibition with nemolizumab and confirm the critical upstream role of IL-31 in PN pathogenesis.

CD4+ T Cells Have a Permissive Effect on Enriched Environment-Induced Hippocampus Synaptic Plasticity.

Living in an enriched environment (EE) benefits health by acting synergistically on various biological systems including the immune and the central nervous systems. The dialog between the brain and the immune cells has recently gained interest and is thought to play a pivotal role in beneficial effects of EE. Recent studies show that T lymphocytes have an important role in hippocampal plasticity, learning, and memory, although the precise mechanisms by which they act on the brain remain elusive. Using a mouse model of EE, we show here that CD4+ T cells are essential for spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. However, CD4+ lymphocytes do not influence EE-induced neurogenesis in the DG of the hippocampus, by contrast to what we previously demonstrated for CD8+ T cells. Importantly, CD4+ T cells located in the choroid plexus have a specific transcriptomic signature as a function of the living environment. Our study highlights the contribution of CD4+ T cells in the brain plasticity and function.

CD8+ T cells are essential for the effects of enriched environment on hippocampus-dependent behavior, hippocampal neurogenesis and synaptic plasticity.

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNFα release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays.

Cytokines produced by both immune and non-immune cells are likely to play roles in the development and/or progression of psychiatric disorders. Indeed, many investigators have compared the blood cytokine levels in psychiatric patients with those of healthy controls or monitored their levels in patients during disease progression to identify biomarkers. Nevertheless, very few studies have confirmed that such cytokines remain stable in healthy individuals through periods of weeks and months. This is an important issue to consider before using blood cytokine levels as biomarkers of disease traits, disease state, or treatment response. Although multiplex assay technology represents an advance in identifying biomarkers because it allows simultaneous examination of large panels of analytes from a small volume of sample, it is necessary to verify whether these assays yield enough sensitivity and reproducibility when applied to the blood from neuropsychiatric patients. Therefore, we compared two multiplex immunoassays, the bead-based Luminex® (Bio-Rad) and the electro-chemiluminescence-based V-plex® (MesoScaleDiscovery), for the detection and quantification of 31 cytokines, chemokines and growth factors in both the sera and plasma of patients with major depressive episodes (MDE) and age- and sex-matched healthy control subjects during a 30-week period. Although both platforms exhibited low coefficients of variability (CV) between the duplicates in the calibration curves, the linearity was better in general for the V-PLEX® platform. However, neither platform was able to detect the absolute values for all of the tested analytes. Among the 16 analytes that were detected by both assays, the intra-assay reproducibility was in general better with the V-PLEX® platform. Although it is not a general rule that the results from sera and plasma will be correlated, consistent results were more frequent with the V-PLEX® platform. Furthermore, the V-PLEX® results were more consistent with the gold standard ELISA simplex assay for IL-6 in both sera and plasma. The intra-individual variability of the measurements, among the sera and plasma for the 4 samples harvested from each healthy individual, was low for Eotaxin, G-CSF, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, MIP-1ß, PDGF-BB, TNF, TNF-ß and VEGF, but intermediate or high for IFN-γ, IL-6, IL-8, IL-10, and IP10. Together, these data suggest that extreme caution is needed in translating the results of multiplex cytokine profiling into biomarker discovery in psychiatry.

Lectin-like transcript 1 is a marker of germinal center-derived B-cell non-Hodgkin's lymphomas dampening natural killer cell functions.

Non-Hodgkin's lymphomas (NHLs) are malignant neoplasms which are clinically and biologically diverse. Their incidence is constantly increasing and despite treatment advances, there is a need for novel targeted therapies. Here, we identified Lectin-like transcript 1 (LLT1) as a biomarker of germinal center (GC)-derived B-cell NHLs. LLT1 identifies GC B cells in reactive tonsils and lymph nodes and its expression is maintained in B-cell NHLs which derive from GC, including Burkitt lymphoma (BL), follicular lymphoma (FL), and GC-derived diffuse large B-cell lymphoma (DLBCL). We further show that LLT1 expression by tumors dampens natural killer (NK) cell functions following interaction with its receptor CD161, uncovering a potential immune escape mechanism. Our results pinpoint LLT1 as a novel biomarker of GC-derived B-cell NHLs and as a candidate target for innovative immunotherapies.

CX3CL1 (fractalkine) and its receptor CX3CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin.

Atopic dermatitis (AD) is a chronic allergic dermatosis characterized by epidermal thickening and dermal inflammatory infiltrates with a dominant Th2 profile during the acute phase, whereas a Th1 profile is characteristic of the chronic stage. Among chemokines and chemokine receptors associated with inflammation, increased levels of CX3CL1 (fractalkine) and its unique receptor, CX3CR1, have been observed in human AD. We have thus investigated their role and mechanism of action in experimental models of AD and psoriasis. AD pathology and immune responses, but not psoriasis, were profoundly decreased in CX3CR1-deficient mice and upon blocking CX3CL1-CX3CR1 interactions in wild-type mice. CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD. Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4(+) T cell retention in inflamed skin, indicating a new function for this chemokine receptor. Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.

Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses.

CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

Splenic CD8α⁺ dendritic cells undergo rapid programming by cytosolic bacteria and inflammation to induce protective CD8⁺ T-cell memory.

Memory CD8(+) T lymphocytes are critical effector cells of the adaptive immune system mediating long-lived pathogen-specific protective immunity. Three signals - antigen, costimulation and inflammation - orchestrate optimal CD8(+) T-cell priming and differentiation into effector and memory cells and shape T-cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α(+) but not the CD8α(-) cDCs most efficiently mediate CD8(+) T-cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I-associated antigens, is most efficient at inducing naïve CD8(+) T-cell differentiation into pathogen-specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α(+) cDCs become endowed with all functional features to optimally prime protective memory CD8(+) T cells in vivo within only a few hours post-immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell-based vaccine therapy.

Priming of protective anti-Listeria monocytogenes memory CD8+ T cells requires a functional SecA2 secretion system.

The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have demonstrated that this secretion system is also linked to the onset of long-term CD8(+) T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8(+) T cells that are required for protection against secondary challenge with wild-type (wt) L. monocytogenes. Since the SecA2 ATPase is well conserved among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3(+) memory CD8(+) T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8(+) T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.

Optimization of a homogeneous assay for kinase inhibitors in plant extracts.

To identify natural and original kinase inhibitors from plant extracts, we have developed and compared a heterogeneous enzyme-linked immunosorbent assay (ELISA) and a homogeneous time-resolved fluorescence (HTRF, Cisbio International, Bagnols/Cèze, France) assay. Kinase affinity for the ATP substrate was determined in both assays, and the same [ATP]/ATP Km ratio was used in each case to enable the identification of ATP competitive and noncompetitive inhibitors. Assays were then used to screen the same collection of chemical compounds and plant extracts. The intra-assay correlation analysis of each technology showed a very good screening precision in HTRF and an acceptable one in ELISA. When the two methods were compared, a poor correlation was obtained with a higher hit rate in the ELISA. We then performed a detailed study of the ELISA hits and showed that they also presented a strong antioxidant activity, associated with high adsorption into microplate wells, which interfered with the horseradish peroxidase-based detection system. These hits were then flagged as false-positives. We also showed that many plant extracts presented this kind of activity and that this interference could explain the lack of correlation between the assays. These findings suggest that assay design should be carefully adapted to the substances to be screened and that interferences should be extensively considered before any assay development process and comparison studies. In spite of a few interferences, our results showed that a homogeneous-phase assay like the HTRF assay could be more efficiently used for plant extract screening than a heterogeneous-phase assay like ELISA.