Characteristics and Clinical Implications of Immunoglobulins Derived from Non B Cells in the Skin.

Skin is the most prominent tissue and organ, as well as the first line of defence, of the body. Because it is situated on the body's surface, it is constantly exposed to microbial, chemical, and physical factors such as mechanical stimulation. Therefore, skin has evolved substantial immune defences, regenerative ability, and anti-injury capacity. Epidermal cells produce antibacterial peptides that play a role in immune defence under physiological conditions. Additionally, IgG or IgA in the skin also participates in local anti-infective immunity. However, based on the classical theory of immunology, Ig can only be produced by B cells which should be derived from local B cells. This year, thanks to the discovery of Ig derived from non B cells (non B-Ig), Ig has also been found to be expressed in epidermal cells and contributes to immune defence. Epidermal cell-derived IgG and IgA have been demonstrated to have potential antibody activity by binding to pathogens. However, these epidermal cell-derived Igs show different microbial binding characteristics. For instance, IgG binds to Staphylococcus aureus and IgA binds to Staphylococcus epidermidis. Epidermal cells producing IgG and IgA may serve as an effective defense mechanism alongside B cells, providing a novel insight into skin immunity.

The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients.

Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). Objective: To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). Methods: To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. Results: We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. Conclusion: The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes in vivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

The MAB-5/Hox family transcription factor is important for Caenorhabditis elegans innate immune response to Staphylococcus epidermidis infection.

Innate immunity functions as a rapid defense against broad classes of pathogenic agents. While the mechanisms of innate immunity in response to antigen exposure are well-studied, how pathogen exposure activates the innate immune responses and the role of genetic variation in immune activity is currently being investigated. Previously, we showed significant survival differences between the N2 and the CB4856 Caenorhabditis elegans isolates in response to Staphylococcus epidermidis infection. One of those differences was expression of the mab-5 Hox family transcription factor, which was induced in N2, but not CB4856, after infection. In this study, we use survival assays and RNA-sequencing to better understand the role of mab-5 in response to S. epidermidis. We found that mab-5 loss-of-function (LOF) mutants were more susceptible to S. epidermidis infection than N2 or mab-5 gain-of-function (GOF) mutants, but not as susceptible as CB4856 animals. We then conducted transcriptome analysis of infected worms and found considerable differences in gene expression profiles when comparing animals with mab-5 LOF to either N2 or mab-5 GOF. N2 and mab-5 GOF animals showed a significant enrichment in expression of immune genes and C-type lectins, whereas mab-5 LOF mutants did not. Overall, gene expression profiling in mab-5 mutants provided insight into MAB-5 regulation of the transcriptomic response of C. elegans to pathogenic bacteria and helps us to understand mechanisms of innate immune activation and the role that transcriptional regulation plays in organismal health.

Engineered skin bacteria induce antitumor T cell responses against melanoma.

Certain bacterial colonists induce a highly specific T cell response. A hallmark of this encounter is that adaptive immunity develops preemptively, in the absence of an infection. However, the functional properties of colonist-induced T cells are not well defined, limiting our ability to understand anticommensal immunity and harness it therapeutically. We addressed both challenges by engineering the skin bacterium Staphylococcus epidermidis to express tumor antigens anchored to secreted or cell-surface proteins. Upon colonization, engineered S. epidermidis elicits tumor-specific T cells that circulate, infiltrate local and metastatic lesions, and exert cytotoxic activity. Thus, the immune response to a skin colonist can promote cellular immunity at a distal site and can be redirected against a target of therapeutic interest by expressing a target-derived antigen in a commensal.

Designer bugs as cancer drugs?

Skin microbiota can be engineered to induce antitumor T cells.

Immune checkpoint inhibitors unleash pathogenic immune responses against the microbiota.

Immune checkpoint inhibitors (ICIs) are essential components of the cancer therapeutic armamentarium. While ICIs have demonstrated remarkable clinical responses, they can be accompanied by immune-related adverse events (irAEs). These inflammatory side effects are of unclear etiology and impact virtually all organ systems, with the most common being sites colonized by the microbiota such as the skin and gastrointestinal tract. Here, we establish a mouse model of commensal bacteria-driven skin irAEs and demonstrate that immune checkpoint inhibition unleashes commensal-specific inflammatory T cell responses. These aberrant responses were dependent on production of IL-17 by commensal-specific T cells and induced pathology that recapitulated the cutaneous inflammation seen in patients treated with ICIs. Importantly, aberrant T cell responses unleashed by ICIs were sufficient to perpetuate inflammatory memory responses to the microbiota months following the cessation of treatment. Altogether, we have established a mouse model of skin irAEs and reveal that ICIs unleash aberrant immune responses against skin commensals, with long-lasting inflammatory consequences.

Interactions between human milk oligosaccharides, microbiota and immune factors in milk of women with and without mastitis.

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women.

Delftia acidovorans secretes substances that inhibit the growth of Staphylococcus epidermidis through TCA cycle-triggered ROS production.

The proportion of Staphylococcus aureus in the skin microbiome is associated with the severity of inflammation in the skin disease atopic dermatitis. Staphylococcus epidermidis, a commensal skin bacterium, inhibits the growth of S. aureus in the skin. Therefore, the balance between S. epidermidis and S. aureus in the skin microbiome is important for maintaining healthy skin. In the present study, we demonstrated that the heat-treated culture supernatant of Delftia acidovorans, a member of the skin microbiome, inhibits the growth of S. epidermidis, but not that of S. aureus. Comprehensive gene expression analysis by RNA sequencing revealed that culture supernatant of D. acidovorans increased the expression of genes related to glycolysis and the tricarboxylic acid cycle (TCA) cycle in S. epidermidis. Malonate, an inhibitor of succinate dehydrogenase in the TCA cycle, suppressed the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis. Reactive oxygen species production in S. epidermidis was induced by the heat-treated culture supernatant of D. acidovorans and suppressed by malonate. Further, the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis was suppressed by N-acetyl-L-cysteine, a free radical scavenger. These findings suggest that heat-resistant substances secreted by D. acidovorans inhibit the growth of S. epidermidis by inducing the production of reactive oxygen species via the TCA cycle.

Cell Immunity in Implant-Associated Infections Caused by Biofilm-Forming Microorganisms.

High biofilm-forming capacity of Staphylococcus spp. strains isolated from biomaterial of patients with infectious complications after primary knee replacement developed within 6-12 months after surgery was experimentally demonstrated. Differential leukocyte counts and some indicators of cell immunity in these patients were compared with those in patients without purulent complications and healthy volunteers. In patients with implant-associated infection, the relative numbers of T cells (both T-helpers and T-suppressors) B cells were significantly (p<0.05) reduced, while the number of NK cells was significantly increased in comparison with the corresponding parameters in other groups. The revealed changes attest to cell immunity failure in biofilm infection.

Role of Interleukin-33 in Staphylococcus epidermidis-Induced Septicemia.

Interleukin (IL)-33 is a member of the IL-1 family, which plays an important role in inflammatory response. In this study, we evaluated the effect of IL-33 on septicemia and the underlying mechanisms by establishing a Staphylococcus epidermidis (S. epidermidis)-induced septicemic mouse model. The expression of IL-33, IL-1α, IL-1ß, IL-6, IL-17A, IL-22, and PGE2 were measured by double antibody sandwich enzyme-linked immunosorbent assay, and bacterial colony formation in peripheral blood and kidneys were counted postinfection. The percentages of neutrophils, eosinophils, and inflammatory monocytes were evaluated by flow cytometry, and tissue damage was assessed by hematoxylin and eosin (H&E) staining. The survival of septicemic mice was monitored daily. IL-33 expression was significantly augmented following S. epidermidis infection. High IL-33 expression significantly decreased the survival of model mice, and aggravated the damage of lung, liver, and kidney tissues. However, administration of ST2 (receptor for IL-33) to the S. epidermidis-infected mice blocked the IL-33 signaling pathway, which elevated PGE2, IL-17A, and IL-22, and promoted healing of organ damage. In addition, ST2 suppressed the mobilization of inflammatory monocytes, but promoted the accumulation of neutrophils and eosinophils in S. epidermidis-infected mice. Inhibition of PGE2, IL-17A, and IL-22 facilitated the development of septicemia and organ damage in S. epidermidis-infected mice, as well as reducing their survival. Our findings reveal that IL-33 aggravates organ damage in septicemic mice by inhibiting PGE2, IL-17A, and IL-22 production.

Symbiotic microbiome Staphylococcus aureus from human nasal mucus modulates IL-33-mediated type 2 immune responses in allergic nasal mucosa.

BACKGROUND: The host-microbial commensalism can shape the innate immune responses in respiratory mucosa and nasal microbiome also modulates front-line immune mechanism in the nasal mucosa. Inhaled allergens encounter the host immune system first in the nasal mucosa, and microbial characteristics of nasal mucus directly impact the mechanisms of initial allergic responses in nasal epithelium. However, the roles of the nasal microbiome in allergic nasal mucosa remain uncertain. We sought to determine the distribution of nasal microbiomes in allergic nasal mucosa and elucidate the interplay between nasal microbiome Staphylococcus species and Th2 cytokines in allergic rhinitis (AR) models. RESULTS: Staphylococcus aureus (AR-SA) and S. epidermidis (AR-SE) were isolated from the nasal mucosa of patients with AR. The influence of nasal microbiome Staphylococcus species on allergic nasal mucosa was also tested with in vitro and in vivo AR models. Pyrosequencing data showed that colonization by S. epidermidis and S. aureus was more dominant in nasal mucus of AR subjects. The mRNA and protein levels of IL-33 and TSLP were significantly higher in AR nasal epithelial (ARNE) cells which were cultured from nasal mucosa of AR subjects, and exposure of ARNE cells to AR-SA reduced IL-33 mRNA and secreted protein levels. Particularly, ovalbumin-driven AR mice inoculated with AR-SA by intranasal delivery exhibited significantly reduced IL-33 in their nasal mucosa. In the context of these results, allergic symptoms and Th2 cytokine levels were significantly downregulated after intranasal inoculation of AR-SA in vivo AR mice. CONCLUSION: Colonization by Staphylococcus species was more dominant in allergic nasal mucosa, and nasal commensal S. aureus from subjects with AR mediates anti-allergic effects by modulating IL-33-dependent Th2 inflammation. The results demonstrate the role of host-bacterial commensalism in shaping human allergic inflammation.

Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin.

Perforin-2 (P-2) is an antimicrobial protein with unique properties to kill intracellular bacteria. Gamma delta (GD) T cells, as the major T cell population in epithelial tissues, play a central role in protective and pathogenic immune responses in the skin. However, the tissue-specific mechanisms that control the innate immune response and the effector functions of GD T cells, especially the cross-talk with commensal organisms, are not very well understood. We hypothesized that the most prevalent skin commensal microorganism, Staphylococcus epidermidis, may play a role in regulating GD T cell-mediated cutaneous responses. We analyzed antimicrobial protein P-2 expression in human skin at a single cell resolution using an amplified fluorescence in situ hybridization approach to detect P-2 mRNA in combination with immunophenotyping. We show that S. epidermidis activates GD T cells and upregulates P-2 in human skin ex vivo in a cell-specific manner. Furthermore, P-2 upregulation following S. epidermidis stimulation correlates with increased ability of skin cells to kill intracellular Staphylococcus aureus. Our findings are the first to reveal that skin commensal bacteria induce P-2 expression, which may be utilized beneficially to modulate host innate immune responses and protect from skin infections.

Microbiota in Rosacea.

Rosacea is a complex facial skin condition associated with abnormal inflammation and vascular dysfunction. Next to the known trigger factors, the role of microbiota in the development and aggravation of rosacea continues to raise interest. Demodex folliculorum mites, Helicobacter pylori, Staphylococcus epidermidis, Chlamydia pneumoniae, and the Demodex-associated bacterium, Bacillus oleronius are microbes that have been linked with rosacea. However, the results of studies which assessed their involvement in the disease have been inconsistent and inconclusive. Microbiological research in many different disciplines exploded in recent years as methods to analyze complex microbial communities at the taxonomic and phylogenetic levels became available. Here, we provide an update on the microorganisms implicated in rosacea and review the potential pathogenic role of microbes in the development of rosacea.

[Reactivity to antigens of the microbiome of the respiratory tract in patients with respiratory allergic diseases]. / Reactividad a antígenos del microbioma de vías respiratorias en pacientes con enfermedad alérgica respiratoria.

BACKGROUND: The prevalence of allergic diseases has increased worldwide. Recent studies have informed that the dysbiosis of some specific members of the human microbiota may enhance the allergic response of the respiratory tract. OBJECTIVE: To retrospectively explore the role of some microorganisms of the human microbiota on the skin reactivity and their effect on the chronicity of allergic respiratory diseases in humans. METHODS: A retrospective analysis of a 5-year database of patients with allergic respiratory tract disease. The frequency and magnitude of the reactivity to 38 different allergens was determined. RESULTS: Dermatophagoides pteronyssinus had the highest frequency of reactivity (93.7 %), followed by the bacterial allergen (a mixture of Staphylococcus aureus and Staphylococcus epidermidis) with a frequency of reactivity of 91.82 %; whereas Candida albicans had a frequency of reactivity of only 79.32 %. The frequency of reactivity to the pollen of native Mexican weeds was even lower ~79 %. CONCLUSION: The microorganisms of the microbiota that were analyzed in this study seem to have an influence on the development of respiratory allergic inflammation, associated with long-term colonization of the pharynx, nasal mucosa, and sinuses because of these microorganisms.

Antecedentes: La prevalencia de las enfermedades alérgicas ha aumentado en todo el mundo. En estudios recientes se ha informado que la disbiosis de algunos miembros específicos de la microbiota humana puede potenciar la respuesta alérgica de las vías respiratorias. Objetivo: Explorar retrospectivamente el papel de algunos microorganismos de la microbiota humana en la reactividad cutánea y su efecto sobre la cronicidad de las enfermedades alérgicas respiratorias en el humano. Métodos: Análisis retrospectivo de la base de datos de un periodo de cinco años de pacientes con enfermedad alérgica de las vías respiratorias. Se determinó la frecuencia y magnitud de la reactividad a 38 alérgenos diferentes. Resultados: La mayor frecuencia de reactividad la presentó Dermatophagoides pteronyssinus (93.7 %), al que le siguió una combinación bacteriana de Staphylococcus aureus-Staphylococcus epidermidis (91.82 %) y Candida albicans (79.32 %). La reactividad a alérgenos de polen de malezas nativas de México fue aun menor, aproximadamente de 79 %. Conclusión: Los microorganismos de la microbiota analizados en este estudio parecen tener una influencia en el desarrollo de la inflamación alérgica respiratoria, asociada a la colonización a largo plazo de la faringe, la mucosa nasal y los senos paranasales.

Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies.

In many natural and clinical settings, bacteria are associated with some type of biotic or abiotic surface that enables them to form biofilms, a multicellular lifestyle with bacteria embedded in an extracellular matrix. Staphylococcus aureus and Staphylococcus epidermidis, the most frequent causes of biofilm-associated infections on indwelling medical devices, can switch between an existence as single free-floating cells and multicellular biofilms. During biofilm formation, cells first attach to a surface and then multiply to form microcolonies. They subsequently produce the extracellular matrix, a hallmark of biofilm formation, which consists of polysaccharides, proteins, and extracellular DNA. After biofilm maturation into three-dimensional structures, the biofilm community undergoes a disassembly process that leads to the dissemination of staphylococcal cells. As biofilms are dynamic and complex biological systems, staphylococci have evolved a vast network of regulatory mechanisms to modify and fine-tune biofilm development upon changes in environmental conditions. Thus, biofilm formation is used as a strategy for survival and persistence in the human host and can serve as a reservoir for spreading to new infection sites. Moreover, staphylococcal biofilms provide enhanced resilience toward antibiotics and the immune response and impose remarkable therapeutic challenges in clinics worldwide. This review provides an overview and an updated perspective on staphylococcal biofilms, describing the characteristic features of biofilm formation, the structural and functional properties of the biofilm matrix, and the most important mechanisms involved in the regulation of staphylococcal biofilm formation. Finally, we highlight promising strategies and technologies, including multitargeted or combinational therapies, to eradicate staphylococcal biofilms.

Modulation of S. epidermidis-induced innate immune responses in neonatal whole blood.

BACKGROUND: Coagulase-negative staphylococci (CoNS) such as Staphylococcus epidermidis are highly prevalent pathogens for sepsis in neonates. The interaction between host, environment and pathogenic factors of S. epidermidis are still poorly understood. Our objective was to address the role of several pathogenic factors of S. epidermidis on neonatal cytokine responses and to characterize the influence of three immunomodulatory drugs. METHODS: We performed an ex-vivo model of S. epidermidis sepsis by assessment of blood cytokine production in neonatal whole blood stimulation assays (ELISA). S. epidermidis strains with different characteristics were added as full pathogen to umbilical cord blood cultures and the influence of indomethacin, ibuprofen and furosemide on neonatal immune response to S. epidermidis was evaluated (Flow cytometry). RESULTS: Stimulation with S. epidermidis sepsis strains induced higher IL-6 and IL-10 expression than stimulation with colonization strains. Biofilm formation in clinical isolates was associated with increased IL-10 but not IL-6 levels. In contrast, stimulation with mutant strains for biofilm formation and extracellular virulence factors had no major effect on cytokine expression. Notably, addition of ibuprofen or indomethacin to S. epidermidis inoculated whole blood resulted in mildly increased expression of TNF-α but not IL-6, while frusemide decreased the production of pro-inflammatory cytokines, i.e. IL-6 and IL-8. CONCLUSIONS: The virulence of sepsis strains is coherent with increased cytokine production in our whole-blood in-vitro sepsis model. Biofilm formation and expression of extracellular virulence factors had no major influence on readouts in our setting. It is important to acknowledge that several drugs used in neonatal care have immunomodulatory potential.