A Bacterial Quorum Sensing Regulated Protease Inhibits Host Immune Responses by Cleaving Death Domains of Innate Immune Adaptors.

Innate immune adaptor proteins are critical components of the innate immune system that propagate pro-inflammatory responses from their upstream receptors, and lead to pathogen clearance from the host. Bacterial pathogens have developed strategies to survive inside host cells without triggering the innate immune surveillance in ways that are still not fully understood. Here, it is reported that Pseudomonas aeruginosa induces its quorum sensing mechanism after macrophage engulfment. Further investigation of its secretome identified a quorum sensing regulated product, LasB, is responsible for innate immune suppression depending on the MyD88-mediated signaling. Moreover, it is showed that this specific type of pathogen-mediated innate immune suppression is due to the enzymatic digestion of the death domains of the innate immune adaptors, mainly MyD88, and attributed to LasB's large substrate binding groove. Lastly, it is demonstrated that the secretion of LasB from P. aeruginosa directly contributed to MyD88 degradation within macrophages. Hence, it is discovered an example of bacterial quorum sensing-regulated cellular innate immune suppression by direct cleavage of immune adaptors.

Age-Related Alterations in Macrophage Distribution and Function Are Associated With Delayed Cutaneous Wound Healing.

Ageing-related delays and dysregulated inflammation in wound healing are well-documented in both human and animal models. However, cellular and molecular changes underlying this impairment in healing progression are not fully understood. In this study, we characterised ageing-associated changes to macrophages in wounds of young and aged mice and investigated transcriptomic differences that may impact the progression of wound healing. Full-thickness wounds created on the dorsum of C57BL/6J young and aged mice were excised on Days 3 and 7 post-wounding for analysis by immunohistochemistry, flow cytometry, and RNA sequencing. Our data revealed that macrophages were significantly reduced in aged wounds in comparison to young. Functional transcriptomic analyses showed that macrophages from aged wounds exhibited significantly reduced expression of cell cycle, DNA replication, and repair pathway genes. Furthermore, we uncovered an elevated pro-inflammatory gene expression program in the aged macrophages correlated with poor inflammation resolution and excessive tissue damage observed in aged wounds. Altogether, our work provides insights into how poorly healing aged wounds are phenotypically defined by the presence of macrophages with reduced proliferative capacity and an exacerbated inflammatory response, both of which are pathways that can be targeted to improve healing in the elderly.

Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

Single-cell analysis of human skin identifies CD14+ type 3 dendritic cells co-producing IL1B and IL23A in psoriasis.

Inflammatory skin diseases including atopic dermatitis (AD) and psoriasis (PSO) are underpinned by dendritic cell (DC)-mediated T cell responses. Currently, the heterogeneous human cutaneous DC population is incompletely characterized, and its contribution to these diseases remains unclear. Here, we performed index-sorted single-cell flow cytometry and RNA sequencing of lesional and nonlesional AD and PSO skin to identify macrophages and all DC subsets, including the newly described mature LAMP3+BIRC3+ DCs enriched in immunoregulatory molecules (mregDC) and CD14+ DC3. By integrating our indexed data with published skin datasets, we generated a myeloid cell universe of DC and macrophage subsets in healthy and diseased skin. Importantly, we found that CD14+ DC3s increased in PSO lesional skin and co-produced IL1B and IL23A, which are pathological in PSO. Our study comprehensively describes the molecular characteristics of macrophages and DC subsets in AD and PSO at single-cell resolution, and identifies CD14+ DC3s as potential promoters of inflammation in PSO.

Autoimmune Pemphigus: Latest Advances and Emerging Therapies.

Pemphigus represents a group of rare and severe autoimmune intra-epidermal blistering diseases affecting the skin and mucous membranes. These painful and debilitating diseases are driven by the production of autoantibodies that are mainly directed against the desmosomal adhesion proteins, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1). The search to define underlying triggers for anti-Dsg-antibody production has revealed genetic, environmental, and possible vaccine-driven factors, but our knowledge of the processes underlying disease initiation and pathology remains incomplete. Recent studies point to an important role of T cells in supporting auto-antibody production; yet the involvement of the myeloid compartment remains unexplored. Clinical management of pemphigus is beginning to move away from broad-spectrum immunosuppression and towards B-cell-targeted therapies, which reduce many patients' symptoms but can have significant side effects. Here, we review the latest developments in our understanding of the predisposing factors/conditions of pemphigus, the underlying pathogenic mechanisms, and new and emerging therapies to treat these devastating diseases.

A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor α (VEGF-α) by a minor subset of activated EpCAM+CD59+Ly-6D+ cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guérin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Ezh2 Controls Skin Tolerance through Distinct Mechanisms in Different Subsets of Skin Dendritic Cells.

Ezh2, a well-established epigenetic repressor, can down-regulate leukocyte inflammatory responses, but its role in cutaneous health remains elusive. Here we demonstrate that Ezh2 controls cutaneous tolerance by regulating Langerhans cell (LC) transmigration across the epidermal basement membrane directly via Talin1 methylation. Ezh2 deficiency impaired disassembly of adhesion structures in LCs, leading to their defective integrin-dependent emigration from the epidermis and failure in tolerance induction. Moreover, mobilization of Ezh2-deficient Langerin- dermal dendritic cells (dDCs) via high-dose treatment with a weak allergen restored tolerance, which is associated with an increased tolerogenic potential of Langerin- dDCs likely due to epigenetic de-repression of Aldh in the absence of Ezh2. Our data reveal novel roles for Ezh2 in governing LC- and dDC-mediated host protection against cutaneous allergen via distinct mechanisms.

Calcineurin-mediated IL-2 production by CD11chighMHCII+ myeloid cells is crucial for intestinal immune homeostasis.

The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1 CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin-NFAT-IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine.

New Insights into Drug Reaction with Eosinophilia and Systemic Symptoms Pathophysiology.

Drug reaction with eosinophilia and systemic symptoms (DRESS), also known as drug-induced hypersensitivity syndrome, is a severe type of cutaneous drug-induced eruption. DRESS may be a difficult disease to diagnose since the symptoms mimic those of cutaneous and systemic infectious pathologies and can appear up to 3 months after the initial culprit drug exposure. The symptoms of DRESS syndrome include rash development after a minimum of 3 weeks after the onset of a new medication, associated with facial edema, lymphadenopathy, and fever. Biological findings include liver abnormalities, leukocytosis, eosinophilia, atypical lymphocytosis, and reactivation of certain human herpes viruses. In DRESS, liver, kidneys, and lungs are frequently involved in disease evolution. Patients with serious systemic involvement are treated with oral corticosteroids, and full recovery is achieved in the majority of cases. DRESS is a rare disease, and little is known about factors that predict its occurrence. The key features of this reaction are eosinophil involvement, the role of the culprit drug, and virus reactivation that trigger an inappropriate systemic immune response in DRESS patients. Interestingly, it was evidenced that at-risk individuals within a genetically restricted population shared a particular HLA loci. In this respect, a limited number of well-known drugs were able to induce DRESS. This review describes the up-to-date advances in our understanding of the pathogenesis of DRESS.

Enterococcus faecalis Modulates Immune Activation and Slows Healing During Wound Infection.

Enterococcus faecalis is one of the most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics of E faecalis and show that infected wounds result in 2 different states depending on the initial inoculum. Low-dose inocula were associated with short-term, low-titer colonization whereas high-dose inocula were associated with acute bacterial replication and long-term persistence. High-dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high-dose infection, the multiple peptide resistance factor, which is involved in resisting immune clearance, contributes to E faecalis fitness. These results comprehensively describe a mouse model for investigating E faecalis wound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, nonhealing wounds.

Human fetal dendritic cells promote prenatal T-cell immune suppression through arginase-2.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Whole metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare.

Whole metagenome analysis has the potential to reveal functional triggers of skin diseases, but issues of cost, robustness and sampling efficacy have limited its application. Here, we have established an alternative, clinically practical and robust metagenomic analysis protocol and applied it to 80 skin microbiome samples epidemiologically stratified for atopic dermatitis (AD). We have identified distinct non-flare, baseline skin microbiome signatures enriched for Streptococcus and Gemella but depleted for Dermacoccus in AD-prone versus normal healthy skin. Bacterial challenge assays using keratinocytes and monocyte-derived dendritic cells established distinct IL-1-mediated, innate and Th1-mediated adaptive immune responses with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial differences were complemented by perturbations in the eukaryotic community and functional shifts in the microbiome-wide gene repertoire, which could exacerbate a dry and alkaline phenotype primed for pathogen growth and inflammation in AD-susceptible skin. These findings provide insights into how the skin microbial community, skin surface microenvironment and immune system cross-modulate each other, escalating the destructive feedback cycle between them that leads to AD flare.

Cdk2 catalytic activity is essential for meiotic cell division in vivo.

Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2(D145N/D145N) or Cdk2(T160A/T160A) expressed only 'kinase-dead' variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only 'kinase-dead' variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals.

Protocols for the Identification and Isolation of Antigen-Presenting Cells in Human and Mouse Tissues.

The efficient processing of both mouse and human tissues is a valuable technique for characterizing tissue-associated immune cells. Here, we describe the techniques used and optimised within our laboratory for the enrichment and identification of antigen-presenting cells across a number of mouse and human tissues.

CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease.

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.

Drug reaction with eosinophilia and systemic symptoms (DRESS): a multiorgan antiviral T cell response.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, drug-induced reaction that involves both the skin and the viscera. Evidence for reactivation of herpes family viruses has been seen in some DRESS patients. To understand the immunological components of DRESS and their relationship to viral reactivation, we prospectively assessed 40 patients exhibiting DRESS in response to carbamazepine, allopurinol, or sulfamethoxazole. Peripheral blood T lymphocytes from the patients were evaluated for phenotype, cytokine secretion, and repertoire of CD4+ and CD8+ and for viral reactivation. We found Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), or HHV-7 reactivation in 76% of the patients. In all patients, circulating CD8+ T lymphocytes were activated, exhibited increased cutaneous homing markers, and secreted large amounts of tumor necrosis factor-alpha and interferon-gamma. The production of these cytokines was particularly high in patients with the most severe visceral involvement. In addition, expanded populations of CD8+ T lymphocytes sharing the same T cell receptor repertoire were detected in the blood, skin, liver, and lungs of patients. Nearly half of these expanded blood CD8+ T lymphocytes specifically recognized one of several EBV epitopes. Finally, we found that the culprit drugs triggered the production of EBV in patients' EBV-transformed B lymphocytes. Thus, cutaneous and visceral symptoms of DRESS are mediated by activated CD8+ T lymphocytes, which are largely directed against herpes viruses such as EBV.