Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation.

Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs.

Delayed antiretroviral therapy in HIV-infected individuals leads to irreversible depletion of skin- and mucosa-resident memory T cells.

People living with HIV (PLWH) are at increased risk for developing skin and mucosal malignancies despite systemic reconstitution of CD4+ T cells upon antiretroviral therapy (ART). The underlying mechanism of chronic tissue-related immunodeficiency in HIV is unclear. We found that skin CD4+ tissue-resident memory T (Trm) cells were depleted after HIV infection and replenished only upon early ART initiation. TCR clonal analysis following early ART suggested a systemic origin for reconstituting CD4+ Trm cells. Single-cell RNA sequencing in PLWH that received late ART treatment revealed a loss of CXCR3+ Trm cells and a tolerogenic skin immune environment. Human papilloma virus-induced precancerous lesion biopsies showed reduced CXCR3+ Trm cell frequencies in the mucosa in PLWH versus HIV- individuals. These results reveal an irreversible loss of CXCR3+ Trm cells confined to skin and mucosa in PLWH who received late ART treatment, which may be a precipitating factor in the development of HPV-related cancer.

Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.

Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.

Aberrant Lipid Metabolism in Macrophages Is Associated with Granuloma Formation in Sarcoidosis.

Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development. Objectives: To understand whether macrophages from patients with nonresolving chronic sarcoidosis are predisposed to macrophage aggregation and granuloma formation and whether modulation of the underlying molecular pathways influence sarcoidosis granuloma formation. Methods: Macrophages were cultivated in vitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses and phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from patients with sarcoidosis and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from patients with chronic sarcoidosis spontaneously formed extensive granulomas in vitro compared with healthy control participants. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both in vitro and in vivo in a sarcoidosis mouse model. Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients.

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. OBJECTIVES: To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. METHODS: Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. RESULTS: We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell-cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. CONCLUSIONS: Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

High cure rates of Mycoplasma genitalium following empiric treatment with azithromycin alongside frequent detection of macrolide resistance in Austria.

BACKGROUND: Mycoplasma genitalium (MG) is an emerging sexually transmitted infection, often harboring resistance-associated mutations to azithromycin (AZM). Global surveillance has been mandated to tackle the burden caused by MG, yet no data are available for Austria. Thus, we aimed to investigate the prevalence of MG, disease characteristics, and treatment outcomes at the largest Austrian HIV-and STI clinic. METHODS: All MG test results at the Medical University of Vienna from 02/2019 to 03/2022 were evaluated. Azithromycin resistance testing was implemented in 03/2021. RESULTS: Among 2671 MG tests, 199 distinct and mostly asymptomatic (68%; 135/199) MG infections were identified, affecting 10% (178/1775) of all individuals. This study included 83% (1479/1775) men, 53% (940/1775) men who have sex with men (MSM), 31% (540/1754) HIV+, and 15% (267/1775) who were using HIV pre-exposure prophylaxis (PrEP). In logistic regression analysis, 'MSM' (aOR 2.55 (95% CI 1.65-3.92)), 'use of PrEP' (aOR 2.29 (95% CI 1.58-3.32)), and 'history of syphilis' (aOR 1.57 (95% CI 1.01-2.24) were independent predictors for MG infections. Eighty-nine percent (178/199) received treatment: 11% (21/178) doxycycline (2 weeks), 52% (92/178) AZM (5 days), and 37% ( 65/178) moxifloxacin (7-10 days) and 60% (106/178) had follow-up data available showing negative tests in 63% (5/8), 76% (44/58) and 85% (34/40), respectively. AZM resistance analysis was available for 57% (114/199)) and detected in 68% (78/114). Resistance-guided therapy achieved a cure in 87% (53/61), yet, empiric AZM-treatment (prior to 03/2021) cleared 68% (26/38). CONCLUSIONS: Mycoplasma genitalium was readily detected in this Austrian observational study, affected predominantly MSM and often presented as asymptomatic disease. We observed a worryingly high prevalence of AZM resistance mutations; however, empiric AZM treatment cleared twice as many MG infections as expected.

Asymptomatic lymphogranuloma venereum is commonly found among men who have sex with men in Austria.

BACKGROUND AND OBJECTIVES: Serovar L1-L3 of Chlamydia trachomatis (CT) causes lymphogranuloma venereum (LGV). A surge in LGV-cases has been observed among HIV-positive men who have sex with men (MSM). Discrimination between LGV and non-LGV is pivotal since it has major treatment implications. Here, we aimed to determine the prevalence and characteristics of LGV among CT-infections. PATIENTS AND METHODS: All CT-positive results from 04/2014-12/2021 at the four largest Austrian HIV and STI clinics were evaluated. Disease characteristics and patient demographics were analyzed. RESULTS: Overall, n  =  2,083 infections of CT were documented in n  =  1,479 individual patients: median age was 31.4 years, 81% were male, 59% MSM, 44% HIV-positive, 13% on HIV pre-exposure-prophylaxis. Available serovar analyses (61% [1,258/2,083]) showed L1-L3 in 15% (192/1,258). Considering only MSM with rectal CT-infection, LGV accounted for 23% (101/439). Cases of LGV vs. other CT-infections were primarily MSM (92% [177/192] vs. 62% [1,179/1,891], p < 0.001), more often HIV-positive (64% [116/180] vs. 46% [631/1,376]; p < 0.001) and had frequently concomitant syphilis infection (18% [32/180] vs. 7% [52/749]; p < 0.001). LGV commonly manifested as proctitis (38% [72/192]) whereas 45% (87/192) were asymptomatic. CONCLUSIONS: Lymphogranuloma venereum accounted for 23% of rectal CT-infections in MSM. Furthermore, 45% of all LGV-cases were asymptomatic. In the absence of CT-serovar analysis, a high LGV prevalence should be considered in risk-populations and guide empiric treatment selection.

Epigenetic regulation of T cell lineages in skin and blood following hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem-cell transplantation (HSCT) seeks to reconstitute the host's immune system from donor stem cells. The success of HSCT is threatened by complications including leukemia relapse or graft-versus-host-disease (GvHD). To investigate the underlying regulatory processes in central and peripheral T cell recovery, we performed sequential multi-omics analysis of T cells of the skin and blood during HSCT. We detected rapid effector T cell reconstitution, while emergence of regulatory T cells was delayed. Epigenetic and gene-regulatory programs were associated with recovering T cells and diverged greatly between skin and blood T cells. The BRG1/BRM-associated factor chromatin remodeling complex and histone deacetylases (HDACs) were epigenetic regulators involved in restoration of T cell homeostasis after transplantation. In isolated T cells of patients after HSCT, we observed class I HDAC-inhibitors to modulate their dysbalance. The present study highlights the importance of epigenetic regulation in the recovery of T cells following HSCT.

Dermatovenerological infections.

In dermatology, infections caused by bacteria, viruses, fungi, and parasites play an important role. A large proportion of pathogen-related infections of the skin and mucous membranes are transmitted sexually. All areas of infectious diseases and dermatovenerology are subject to highly exciting, dynamic change. This is driven by changes in the epidemiology of long-established diseases, changes in the resistance of pathogens to anti-infectives, recurrence of known pathogens, and the emergence of completely new pathogens. In this article, we address "resistance to anti-infectives", "sexually transmitted infections", and "emerging viral infections", three core areas of dermatovenerology that will shape the field in the years to come.

Syphilis.

Syphilis is a curable systemic infectious disease with a clear increase in incidence in recent years. The disease presents with a broad clinical spectrum and challenges clinicians due to the long incubation period and the sometimes complex interpretation of serological test results. Penicillin G remains the treatment of choice in all stages of syphilis.

Distinct Chemokine Receptor Expression Profiles in De Novo DLBCL, Transformed Follicular Lymphoma, Richter's Trans-Formed DLBCL and Germinal Center B-Cells.

Chemokine receptors and their ligands have been identified as playing an important role in the development of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Richter syndrome (RS). Our aim was to investigate the different expression profiles in de novo DLBCL, transformed follicular lymphoma (tFL), and RS. Here, we profiled the mRNA expression levels of 18 chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, CX3CR1 and XCR1) using RQ-PCR, as well as immunohistochemistry of seven chemokine receptors (CCR1, CCR4-CCR8 and CXCR2) in RS, de novo DLBCL, and tFL biopsy-derived tissues. Tonsil-derived germinal center B-cells (GC-B) served as non-neoplastic controls. The chemokine receptor expression profiles of de novo DLBCL and tFL substantially differed from those of GC-B, with at least 5-fold higher expression of 15 out of the 18 investigated chemokine receptors (CCR1-CCR9, CXCR1, CXCR2, CXCR6, CXCR7, CX3CR1 and XCR1) in these lymphoma subtypes. Interestingly, the de novo DLBCL and tFL exhibited at least 22-fold higher expression of CCR1, CCR5, CCR8, and CXCR6 compared with RS, whereas no significant difference in chemokine receptor expression profile was detected when comparing de novo DLBCL with tFL. Furthermore, in de novo DLBCL and tFLs, a high expression of CCR7 was associated with a poor overall survival in our study cohort, as well as in an independent patient cohort. Our data indicate that the chemokine receptor expression profile of RS differs substantially from that of de novo DLBCL and tFL. Thus, these multiple dysregulated chemokine receptors could represent novel clinical markers as diagnostic and prognostic tools. Moreover, this study highlights the relevance of chemokine signaling crosstalk in the tumor microenvironment of aggressive lymphomas.

Alum triggers infiltration of human neutrophils ex vivo and causes lysosomal destabilization and mitochondrial membrane potential-dependent NET-formation.

Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so-called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)-adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET-formation. Moreover, we characterized the mechanism leading to alum-induced NET-release in human neutrophils as rapid, NADPH oxidase-independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+ -flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET-release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum-containing vaccines in humans and provides novel insights into bioenergetic requirements of NET-formation.

BMP7 aberrantly induced in the psoriatic epidermis instructs inflammation-associated Langerhans cells.

BACKGROUND: Epidermal hyperplasia represents a morphologic hallmark of psoriatic skin lesions. Langerhans cells (LCs) in the psoriatic epidermis engage with keratinocytes (KCs) in tight physical interactions; moreover, they induce T-cell-mediated immune responses critical to psoriasis. OBJECTIVE: This study sought to improve the understanding of epidermal factors in psoriasis pathogenesis. METHODS: BMP7-LCs versus TGF-ß1-LCs were phenotypically characterized and their functional properties were analyzed using flow cytometry, cell kinetic studies, co-culture with CD4 T cells, and cytokine measurements. Furthermore, immunohistology of healthy and psoriatic skin was performed. Additionally, in vivo experiments with Junf/fJunBf/fK5cre-ERT mice were carried out to assess the role of bone morphogenetic protein (BMP) signaling in psoriatic skin inflammation. RESULTS: This study identified a KC-derived signal (ie, BMP signaling) to promote epidermal changes in psoriasis. Whereas BMP7 is strictly confined to the basal KC layer in the healthy skin, it is expressed at high levels throughout the lesional psoriatic epidermis. BMP7 instructs precursor cells to differentiate into LCs that phenotypically resemble psoriatic LCs. These BMP7-LCs exhibit proliferative activity and increased sensitivity to bacterial stimulation. Moreover, aberrant high BMP signaling in the lesional epidermis is mediated by a KC intrinsic mechanism, as suggested from murine data and clinical outcome after topical antipsoriatic treatment in human patients. CONCLUSIONS: These data indicate that available TGF-ß family members within the lesional psoriatic epidermis preferentially signal through the canonical BMP signaling cascade to instruct inflammatory-type LCs and to promote psoriatic epidermal changes. Targeting BMP signaling might allow to therapeutically interfere with cutaneous psoriatic manifestations.

COVID-19 and immunological regulations - from basic and translational aspects to clinical implications.

The COVID-19 pandemic caused by SARS-CoV-2 has far-reaching direct and indirect medical consequences. These include both the course and treatment of diseases. It is becoming increasingly clear that infections with SARS-CoV-2 can cause considerable immunological alterations, which particularly also affect pathogenetically and/or therapeutically relevant factors. Against this background we summarize here the current state of knowledge on the interaction of SARS-CoV-2/COVID-19 with mediators of the acute phase of inflammation (TNF, IL-1, IL-6), type 1 and type 17 immune responses (IL-12, IL-23, IL-17, IL-36), type 2 immune reactions (IL-4, IL-13, IL-5, IL-31, IgE), B-cell immunity, checkpoint regulators (PD-1, PD-L1, CTLA4), and orally druggable signaling pathways (JAK, PDE4, calcineurin). In addition, we discuss in this context non-specific immune modulation by glucocorticosteroids, methotrexate, antimalarial drugs, azathioprine, dapsone, mycophenolate mofetil and fumaric acid esters, as well as neutrophil granulocyte-mediated innate immune mechanisms. From these recent findings we derive possible implications for the therapeutic modulation of said immunological mechanisms in connection with SARS-CoV-2/COVID-19. Although, of course, the greatest care should be taken with patients with immunologically mediated diseases or immunomodulating therapies, it appears that many treatments can also be carried out during the COVID-19 pandemic; some even appear to alleviate COVID-19.