Evaluating IC31-induced cellular and mucosal humoral immune protective mechanisms against intracellular bacterial infections using a well established Chlamydia pneumoniae model

Chlamydia pneumoniae ist ein generell weit verbreitetes, bakterielles, humanes Pathogen der Atemwege und verursacht ein- oder mehrmals pro Lebenszeit Infektionen bei der Mehrheit aller Menschen. Behandlungen von akuten und persistierenden Cpn Infektionen durch intensive und lang anhaltende Verabreichung von Antibiotika haben sich als effektiv erwiesen. Die meisten Infektionen verlaufen jedoch asymtomatisch oder in Form subklinischer Erkrankungen. Besonders persistierende Cpn Infektionen wurden in Zusam-menhang mit chronischen inflammatorischen Erkrankungen der Atemwege sowie kardiovaskulären Erkrankrungen gebracht. Zusätzlich haben in vitro Studien gezeigt, dass Chlamydien Antibiotikaresistenzen entwickeln können. Gemeinsam unterstützen diese Tatsachen die Wichtigkeit und Vorteilhaftigkeit eines prophylaktischen und therapeutischen Impfstoffes, welcher sowohl vor akuten als auch vor chronischen Cpn Erkrankungen schützt. Die bewiesene zentrale Rolle von CD8+ T Zellen, aber auch CD4+ T Zellen und der humoralen Immunantwort vom Typ TH1, verlangt eine leistungsfähige Stimulation der entsprechenden Effektormechanismen um bei der Entwicklung eines Impfstoffes gegen Cpn erfolgreich zu sein. Für das aus zwei Komponenten bestehende Adjuvans IC31® wurde bereits gezeigt, dass dieses ein wirkungsvoller Stimulator von CD8+ cytotoxischen T Lymphocyten (CTL) und CD4+ T Helfer (Th) Immunantworten in Folge systemischer Verabreichung von T-Zell Antigenen ist. Des Weiteren wurde für IC31® die Effizienz des adjuvantiven Effekts in der mukosalen Anwendung, unter Verwendung des protektiven Cpn Antigens CopN, bewiesen. Weitere vielversprechende Protein Antigene wurden im Verlauf des AIP® (Antigen-Identifizierungs-Programm) unter Verwendung von humanen Seren, welche von infizierten oder von Krankheit regenerierten Patienten stammen, identifiziert. Während dieser Studie wurden die durch IC31® induzierten zellulären sowie humoralen Immunantworten gegen sechs im Prozess des AIP® identifizierten, konservierten, rekombinanten Cpn Antigenen in Immunisierungsstudien und Protektionsstudien evaluiert. Zusätzlich wurde die Relevanz dieser Antigene im Verlauf einer Cpn Infektion, hinsichtlich zellulärer und humoraler Immunogenität, in durch Infektion angeregten Immunantworten von BALB/c und C57BL/6 Mäusen, untersucht. Außerdem konnte durch die Anwendung von Immunofluoreszenzmikroskopie und der Verwendung von aus immunisierten Mäusen stammender Seren die Lokalisation der besagten Antigene/Proteine bestimmt werden. Diese Studie hat gezeigt, dass das Adjuvans IC31® eine überaus wirkvolle Induktion von antigenspezifischen zellulären und humoralen Immun-antworten bewirkt und dies sowohl bei mukosaler als auch systemischer Verabreichung. Des Weiteren unterstützen die Ergebnisse die dominante Induktion einer Immunantwort vom Typ TH1 in einer gemischten TH1/TH2 Immunantwort durch IC31®. Zusätzlich konnten aus den 6 Kandidaten 2 identifiziert werden welche einen substanziellen, reduzierenden Effekt auf die Anzahl an Bakterien in den Lungen von infizierten Mäusen aufwiesen, wenn diese zuvor in Kombination mit dem Adjuvant IC31® intra nasal verabreicht wurden. Diese Kandidaten sind CP0177-1 und CP0020-1. Des Weitern konnten immunogene Epitope dieser beiden Proteine in immunisierten und infizierten BALB/c und C57BL/6 Mäusen durch IFN-γ ELIspot und CFSE-CD3+-CD4+ durchflusscytometrische Analysen identifiziert werden. Zusammengefasst zeigen die Ergebnisse einerseits das große Potential des Adjuvanten IC31® in mukosalen Anwendungen oder als Bestandteil nasaler Impfstoffe und andererseits die hohe Effizienz des AIP® beim Auffinden neuartiger protektiver Impfstoffkandidaten.Chlamydia pneumoniae (Cpn) is a generally widely distributed respiratory human pathogen causing infections in the great majority of individuals once in a lifetime. Intensive and long lasting use of antibiotics is efficient in treating Cpn acute and persistent infections. Though, the majority of infections are asymptomatic or subclinical and especially persistent infection has been associated with a variety of chronic respiratory tract inflammatory diseases and cardiovascular diseases. Furthermore the development of antibiotic resistances has been observed in vitro which underlines the importance and advantages of a future prophylactic and therapeutic vaccine against acute and chronic Cpn infections. Given the essential role of TH1-type immune responses, primarily CD8+ T cells and CD4+ T cells but also humoral responses, in immunity to Cpn, potent induction of respective immune effector mechanisms is a requirement for successful vaccine design. The two-component adjuvant IC31® has been shown to efficiently induce CD8+ cytotoxic T lymphocyte (CTL) and CD4+ T helper (Th) responses to T cell antigens following their systemic administration. Additionally the efficacy of mucosal adjuvanticity following intra nasal immunization was demonstrated for IC31® using the protective Cpn antigen CopN. Further promising Cpn candidate antigens have been identified through the Antigen Identification Program (AIP®) by antibodies from infected/ exposed humans. During this study IC31®-induced cellular and humoral immune responses to 6 AIP®-discovered conserved recombinant Cpn antigens were evaluated in immunization and protection studies. Furthermore, the relevance of these antigens in Cpn infection in terms of cellular and humoral immunogenicity was assessed in infection primed immune response studies using BALB/c and C57BL/6 mice. Moreover, by applying immuno-fluorescence microscopy the 6 candidate antigens could be located using sera from immunized animals. This study demonstrated the efficacy of IC31® in the induction of cellular and humoral responses in mucosal as well as systemic applications, supporting previous findings of a dominant TH1-type in a mixed TH1/TH2-type induction of immune responses. In addition two out of the 6 antigen candidates, namely CP0177-1 and CP0020-1, were found to substantially reduce the bacterial burden in Cpn challenged mice following intra nasal immunization in combination with IC31®. Moreover immunogenic epitopes of these two antigens were identified by IFN-γ ELIspot and CFSE-CD3+-CD4+ flow cyto-metry in protein immunized and infection primed BALB/c and C57BL/6 mice using peptide libraries. All together the results demonstrate the high potential of IC31® as a mucosal adjuvant and its application in nasal vaccines, as well as the efficiency of the AIP® technology for the identification of novel subunit vaccine candidates

ERK phosphorylation and miR-181a expression modulate activation of human memory TH17 cells

T helper (TH) cell polarization during priming is modulated by a number of signals, but whether polarization to a given phenotype also influences recall responses of memory TH cells is relatively unknown. Here we show that miR-181a is selectively induced in both human and mouse naive T cells differentiating into the TH17, but not TH1 or TH2 subset. In human memory TH17 cells, miR-181a regulates responses to cognate antigens through modulation of ERK phosphorylation. By enhancing the signalling cascade from the T-cell receptor, such molecular network reduces the threshold of TH17 cell activation. Moreover, at a late time point, the same network induces a self-regulatory mechanism dependent on ID3, a negative regulator of transcription factors that control RORC expression, thus modulating TH17 activity. Our results demonstrate that the phenotype acquired by TH cells during priming contributes to their threshold of activation to secondary antigenic stimulations, thus influencing memory responses

Deconvolution of monocyte responses in inflammatory bowel disease reveals an IL-1 cytokine network that regulates IL-23 in genetic and acquired IL-10 resistance.

OBJECTIVE: Dysregulated immune responses are the cause of IBDs. Studies in mice and humans suggest a central role of interleukin (IL)-23-producing mononuclear phagocytes in disease pathogenesis. Mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies as part of personalised medicine. DESIGN: We performed transcriptomic analysis to investigate IL-23 expression in human mononuclear phagocytes and peripheral blood mononuclear cells. We investigated the regulation of IL-23 expression and used single-cell RNA sequencing to derive a transcriptomic signature of hyperinflammatory monocytes. Using gene network correlation analysis, we deconvolved this signature into components associated with homeostasis and inflammation in patient biopsy samples. RESULTS: We characterised monocyte subsets of healthy individuals and patients with IBD that express IL-23. We identified autosensing and paracrine sensing of IL-1α/IL-1β and IL-10 as key cytokines that control IL-23-producing monocytes. Whereas Mendelian genetic defects in IL-10 receptor signalling induced IL-23 secretion after lipopolysaccharide stimulation, whole bacteria exposure induced IL-23 production in controls via acquired IL-10 signalling resistance. We found a transcriptional signature of IL-23-producing inflammatory monocytes that predicted both disease and resistance to antitumour necrosis factor (TNF) therapy and differentiated that from an IL-23-associated lymphocyte differentiation signature that was present in homeostasis and in disease. CONCLUSION: Our work identifies IL-10 and IL-1 as critical regulators of monocyte IL-23 production. We differentiate homeostatic IL-23 production from hyperinflammation-associated IL-23 production in patients with severe ulcerating active Crohn's disease and anti-TNF treatment non-responsiveness. Altogether, we identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1β-targeting therapies upstream of IL-23

Immunodeficiency, autoimmune thrombocytopenia and enterocolitis caused by autosomal recessive deficiency of PIK3CD-encoded phosphoinositide 3-kinase δ.

Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase consisting of a catalytic (p110δ, encoded by PIK3CD) and a regulatory subunit (p85, encoded by PIK3R1), generates the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane of leukocytes downstream of antigen and cytokine receptors.1 Signaling via PDK1, AKT, mTOR and downstream targets such as FOXO1, contributes to the metabolic and transcriptional changes required for the expansion, differentiation and effector function of lymphocytes. Activating germline mutations in PIK3CD cause the immune dysregulatory disease activated PI3Kδ syndrome (APDS), usually presenting with recurrent sinopulmonary infections in childhood, herpesvirus infections and CD4+ lymphopenia, underscoring the important role of balanced p110δ activity in human adaptive immunity. Ablation of p110δ in mice leads to aberrant T cell responses and intestinal inflammation. In humans, immune dysregulation including severe colitis is present in many cancer patients who are treated with the p110δ-specific inhibitor Idelalisib. Recently, one patient with autosomal recessive deficiency of p85α and two patients with loss-of function mutations in p110δ have been described who developed humoral immunodeficiency and colitis

Hermansky-Pudlak syndrome type 1 causes impaired anti-microbial immunity and inflammation due to dysregulated immunometabolism

Hermansky-Pudlak syndrome (HPS) types 1 and 4 are caused by defective vesicle trafficking. The mechanism for Crohn's disease-like inflammation, lung fibrosis, and macrophage lipid accumulation in these patients remains enigmatic. The aim of this study is to understand the cellular basis of inflammation in HPS-1. We performed mass cytometry, proteomic and transcriptomic analyses to investigate peripheral blood cells and serum of HPS-1 patients. Using spatial transcriptomics, granuloma-associated signatures in the tissue of an HPS-1 patient with granulomatous colitis were dissected. In vitro studies were conducted to investigate anti-microbial responses of HPS-1 patient macrophages and cell lines. Monocytes of HPS-1 patients exhibit an inflammatory phenotype associated with dysregulated TNF, IL-1α, OSM in serum, and monocyte-derived macrophages. Inflammatory macrophages accumulate in the intestine and granuloma-associated macrophages in HPS-1 show transcriptional signatures suggestive of a lipid storage and metabolic defect. We show that HPS1 deficiency leads to an altered metabolic program and Rab32-dependent amplified mTOR signaling, facilitated by the accumulation of mTOR on lysosomes. This pathogenic mechanism translates into aberrant bacterial clearance, which can be rescued with mTORC1 inhibition. Rab32-mediated mTOR signaling acts as an immuno-metabolic checkpoint, adding to the evidence that defective bioenergetics can drive hampered anti-microbial activity and contribute to inflammation

Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases

Dominant-negative mutations in human IL6ST underlie hyper-IgE syndrome

Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative (DN) STAT3 mutations. Patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, severe allergy, and skeletal abnormalities. We report 12 patients from 8 unrelated kindreds with AD-HIES due to DN IL6ST mutations. We identified seven different truncating mutations, one of which was recurrent. The mutant alleles encode GP130 receptors bearing the transmembrane domain but lacking both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the mutant proteins accumulate at the cell surface and are loss of function and DN for cellular responses to IL-6, IL-11, LIF, and OSM. Moreover, the patients’ heterozygous leukocytes and fibroblasts respond poorly to IL-6 and IL-11. Consistently, patients with STAT3 and IL6ST mutations display infectious and allergic manifestations of IL-6R deficiency, and some of the skeletal abnormalities of IL-11R deficiency. DN STAT3 and IL6ST mutations thus appear to underlie clinical phenocopies through impairment of the IL-6 and IL-11 response pathways

NCX1 represents an ionic Na+ sensing mechanism in macrophages

Inflammation and infection can trigger local tissue Na(+)accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (M phi s) and their antimicrobial activity. Enhanced Na+-driven M phi function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na(+)sensing in M phi s remained unclear. High extracellular Na(+)levels (high salt [HS]) trigger a substantial Na(+)influx and Ca(2+)loss. Here, we show that the Na+/Ca(2+)exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na(+)influx, concomitant Ca(2+)efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na(+)and is required for amplifying inflammatory and antimicrobial M phi responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate M phi function

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19