MPYS Modulates Fatty Acid Metabolism and Immune Tolerance at Homeostasis Independent of Type I IFNs.

MPYS/STING (stimulator of IFN genes) senses cyclic dinucleotides (CDNs), generates type I IFNs, and plays a critical role in infection, inflammation, and cancer. In this study, analyzing genotype and haplotype data from the 1000 Genomes Project, we found that the R71H-G230A-R293Q (HAQ) MPYS allele frequency increased 57-fold in East Asians compared with sub-Saharan Africans. Meanwhile, the G230A-R293Q (AQ) allele frequency decreased by 98% in East Asians compared with sub-Saharan Africans. We propose that the HAQ and AQ alleles underwent a natural selection during the out-of-Africa migration. We used mouse models of HAQ and AQ to investigate the underlying mechanism. We found that the mice carrying the AQ allele, which disappeared in East Asians, had normal CDN-type I IFN responses. Adult AQ mice, however, had less fat mass than did HAQ or wild-type mice on a chow diet. AQ epididymal adipose tissue had increased regulatory T cells and M2 macrophages with protein expression associated with enhanced fatty acid oxidation. Conditional knockout mice and adoptive cell transfer indicate a macrophage and regulatory T cell-intrinsic role of MPYS in fatty acid metabolism. Mechanistically, AQ/IFNAR1-/- mice had a similar lean phenotype as for the AQ mice. MPYS intrinsic tryptophan fluorescence revealed that the R71H change increased MPYS hydrophilicity. Lastly, we found that the second transmembrane (TM) and the TM2-TM3 linker region of MPYS interact with activated fatty acid, fatty acyl-CoA. In summary, studying the evolution of the human MPYS gene revealed an MPYS function in modulating fatty acid metabolism that may be critical during the out-of-Africa migration.

cGAS-STING and MyD88 Pathways Synergize in Ly6Chi Monocyte to Promote Streptococcus pneumoniae-Induced Late-Stage Lung IFNγ Production.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses DNA and induces type I interferon (IFN) production. Whether and how the STING pathway crosstalk to other innate immune pathways during pathogen infection, however, remains unclear. Here, we showed that STING was needed for Streptococcus pneumoniae-induced late, not early, stage of lung IFNγ production. Using knockout mice, IFNγ reporter mice, intracellular cytokine staining, and adoptive cell transfer, we showed that cGAS-STING-dependent lung IFNγ production was independent of type I IFNs. Furthermore, STING expression in monocyte/monocyte-derived cells governed IFNγ production in the lung via the production of IL-12p70. Surprisingly, DNA stimulation alone could not induce IL-12p70 or IFNγ in Ly6Chi monocyte. The production of IFNγ required the activation by both DNA and heat-killed S. pneumococcus. Accordingly, MyD88-/- monocyte did not generate IL-12p70 or IFNγ. In summary, the cGAS-STING pathway synergizes with the MyD88 pathway in monocyte to promote late-stage lung IFNγ production during pulmonary pneumococcal infection.

In vivo reprogramming of pathogenic lung TNFR2+ cDC2s by IFNß inhibits HDM-induced asthma.

Asthma is a common inflammatory lung disease with no known cure. Previously, we uncovered a lung TNFR2+ conventional DC2 subset (cDC2s) that induces regulatory T cells (Tregs) maintaining lung tolerance at steady state but promotes TH2 response during house dust mite (HDM)-induced asthma. Lung IFNß is essential for TNFR2+ cDC2s-mediated lung tolerance. Here, we showed that exogenous IFNß reprogrammed TH2-promoting pathogenic TNFR2+ cDC2s back to tolerogenic DCs, alleviating eosinophilic asthma and preventing asthma exacerbation. Mechanistically, inhaled IFNß, not IFNα, activated ERK2 signaling in pathogenic lung TNFR2+ cDC2s, leading to enhanced fatty acid oxidation (FAO) and lung Treg induction. Last, human IFNß reprogrammed pathogenic human lung TNFR2+ cDC2s from patients with emphysema ex vivo. Thus, we identified an IFNß-specific ERK2-FAO pathway that might be harnessed for DC therapy.

Monocyte-Derived Dendritic Cells (moDCs) Differentiate into Bcl6+ Mature moDCs to Promote Cyclic di-GMP Vaccine Adjuvant-Induced Memory TH Cells in the Lung.

Induction of lung mucosal immune responses is highly desirable for vaccines against respiratory infections. We recently showed that monocyte-derived dendritic cells (moDCs) are responsible for lung IgA induction. However, the dendritic cell subset inducing lung memory TH cells is unknown. In this study, using conditional knockout mice and adoptive cell transfer, we found that moDCs are essential for lung mucosal responses but are dispensable for systemic vaccine responses. Next, we showed that mucosal adjuvant cyclic di-GMP differentiated lung moDCs into Bcl6+ mature moDCs promoting lung memory TH cells, but they are dispensable for lung IgA production. Mechanistically, soluble TNF mediates the induction of lung Bcl6+ moDCs. Our study reveals the functional heterogeneity of lung moDCs during vaccination and paves the way for an moDC-targeting vaccine strategy to enhance immune responses on lung mucosa.

Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD.

Although activation of adaptive immunity is a common pathological feature of chronic obstructive pulmonary disease (COPD), particularly during later stages of the disease, the underlying mechanisms are poorly understood. In small airways of COPD patients, we found that localized disruption of the secretory immunoglobulin A (SIgA)-containing mucosal immunobarrier correlated with lymphocyte accumulation in airway walls and development of tertiary lymphoid structures (TLS) around small airways. In SIgA-deficient mice, we observed bacterial invasion into the airway epithelial barrier with lymphocytic infiltration and TLS formation, which correlated with the progression of COPD-like pathology with advanced age. Depletion of either CD4+ or CD8+ T lymphocytes reduced the severity of emphysema in SIgA-deficient mice, indicating that adaptive immune activation contributes to progressive lung destruction. Further studies revealed that lymphocyte infiltration into the lungs of SIgA-deficient mice was dependent on monocyte-derived dendritic cells (moDCs), which were recruited through a CCR2-dependent mechanism in response to airway bacteria. Consistent with these results, we found that moDCs were increased in lungs of COPD patients, along with CD4+ and CD8+ effector memory T cells. Together, these data indicate that endogenous bacteria in SIgA-deficient airways orchestrate a persistent and pathologic T lymphocyte response through monocyte recruitment and moDC differentiation.

New MoDC-Targeting TNF Fusion Proteins Enhance Cyclic Di-GMP Vaccine Adjuvanticity in Middle-Aged and Aged Mice.

Cyclic dinucleotides (CDNs) are promising vaccine adjuvants inducing balanced, potent humoral, and cellular immune responses. How aging influences CDN efficacy is unclear. We examined the vaccine efficacy of 3',5'-cyclic diguanylic acid (cyclic di-GMP, CDG), the founding member of CDNs, in 1-year-old (middle-aged) and 2-year-old (aged) C57BL/6J mice. We found that 1- and 2-year-old C57BL/6J mice are defective in CDG-induced memory T helper (Th)1 and Th17 responses and high-affinity serum immunoglobulin (Ig)G, mucosal IgA production. Next, we generated two novel tumor necrosis factor (TNF) fusion proteins that target soluble TNF (solTNF) and transmembrane TNF (tmTNF) to monocyte-derived dendritic cells (moDCs) to enhance CDG vaccine efficacy in 1- and 2-year-old mice. The moDC-targeting TNF fusion proteins restored CDG-induced memory Th1, Th17, and high-affinity IgG, IgA responses in the 1- and 2-year-old mice. Together, the data suggested that aging negatively impacts CDG vaccine adjuvanticity. MoDC-targeting TNF fusion proteins enhanced CDG adjuvanticity in the aging mice.

The Age of Cyclic Dinucleotide Vaccine Adjuvants.

As prophylactic vaccine adjuvants for infectious diseases, cyclic dinucleotides (CDNs) induce safe, potent, long-lasting humoral and cellular memory responses in the systemic and mucosal compartments. As therapeutic cancer vaccine adjuvants, CDNs induce potent anti-tumor immunity, including cytotoxic T cells and NK cells activation that achieve durable regression in multiple mouse models of tumors. Clinical trials are ongoing to fulfill the promise of CDNs (ClinicalTrials.gov: NCT02675439, NCT03010176, NCT03172936, and NCT03937141). However, in October 2018, the first clinical data with Merck's CDN MK-1454 showed zero activity as a monotherapy in patients with solid tumors or lymphomas (NCT03010176). Lately, the clinical trial from Aduro's CDN ADU-S100 monotherapy was also disappointing (NCT03172936). The emerging hurdle in CDN vaccine development calls for a timely re-evaluation of our understanding on CDN vaccine adjuvants. Here, we review the status of CDN vaccine adjuvant research, including their superior adjuvant activities, in vivo mode of action, and confounding factors that affect their efficacy in humans. Lastly, we discuss the strategies to overcome the hurdle and advance promising CDN adjuvants in humans.

Lung IFNAR1hi TNFR2+ cDC2 promotes lung regulatory T cells induction and maintains lung mucosal tolerance at steady state.

The lung is a naturally tolerogenic organ. Lung regulatory T cells (T-regs) control lung mucosal tolerance. Here, we identified a lung IFNAR1hiTNFR2+ conventional DC2 (iR2D2) population that induces T-regs in the lung at steady state. Using conditional knockout mice, adoptive cell transfer, receptor blocking antibodies, and TNFR2 agonist, we showed that iR2D2 is a lung microenvironment-adapted dendritic cell population whose residence depends on the constitutive TNFR2 signaling. IFNß-IFNAR1 signaling in iR2D2 is necessary and sufficient for T-regs induction in the lung. The Epcam+CD45- epithelial cells are the sole lung IFNß producer at the steady state. Surprisingly, iR2D2 is plastic. In a house dust mite model of asthma, iR2D2 generates lung TH2 responses. Last, healthy human lungs have a phenotypically similar tolerogenic iR2D2 population, which became pathogenic in lung disease patients. Our findings elucidate lung epithelial cells IFNß-iR2D2-T-regs axis in controlling lung mucosal tolerance and provide new strategies for therapeutic interventions.

Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

Immature lung TNFR2- conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo.

Cyclic dinucleotides (CDNs), including cyclic di-GMP (CDG), are promising vaccine adjuvants in preclinical/clinical trials. The in vivo mechanisms of CDNs are not clear. Here we investigated the roles of lung DC subsets in promoting CDG mucosal adjuvant responses in vivo. Using genetically modified mice and adoptive cell transfer, we identified lung conventional DC 2 (cDC2) as the central player in CDG mucosal responses. We further identified two functionally distinct lung cDC2 subpopulations: TNFR2+pRelB+ and TNFR2-pRelB- cDC2. The TNFR2+ cDC2 were mature and migratory upon intranasal CDG administration while the TNFR2- cDC2 were activated but not mature. Adoptive cell transfer showed that TNFR2- cDC2 mediate the antibody responses of CDG, while the TNFR2+ cDC2 generate Th1/17 responses. Mechanistically, immature TNFR2- cDC2 activate monocyte-derived DCs (moDCs), which do not take up intranasally administered CDG. moDCs promote CDG-induced generation of T follicular helper- and germinal center B cells in the lungs. Our data revealed a previously undescribed in vivo mode of DCs action, whereby an immature lung TNFR2- cDC2 subpopulation directs the non-migratory moDCs to generate CDG mucosal responses in the lung.

Evaluation of Mucosal and Systemic Vaccine Responses by Cyclic di-GMP (CDG)-adjuvanted Protein Subunit Vaccines.

Intranasal administration of vaccine adjuvants directly deliver therapeutic agents to the lungs to induce potent lung mucosal immune responses. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant candidate capable of inducing protective immunity. This protocol describes an in vivo approach to induce and detect mucosal (lung) and systemic (blood and spleen) vaccine adjuvant responses of CDG. This protocol also includes the methods to detect both humoral and cellular immune responses of CDG adjuvant. Last, this protocol can be used to study other cyclic dinucleotides as mucosal vaccine adjuvants.

The Common R71H-G230A-R293Q Human TMEM173 Is a Null Allele.

TMEM173 encodes MPYS/STING and is an innate immune sensor for cyclic dinucleotides (CDNs) playing a critical role in infection, inflammation, and cancer. The R71H-G230A-R293Q (HAQ) of TMEM173 is the second most common human TMEM173 allele. In this study, using data from the 1000 Genomes Project we found that homozygous HAQ individuals account for ∼16.1% of East Asians and ∼2.8% of Europeans whereas Africans have no homozygous HAQ individuals. Using B cells from homozygous HAQ carriers, we found, surprisingly, that HAQ/HAQ carriers express extremely low MPYS protein and have a decreased TMEM173 transcript. Consequently, the HAQ/HAQ B cells do not respond to CDNs. We subsequently generated an HAQ knock-in mouse expressing a mouse equivalent of the HAQ allele (mHAQ). The mHAQ mouse has decreased MPYS protein in B cells, T cells, Ly6Chi monocytes, bone marrow-derived dendritic cells, and lung tissue. The mHAQ mouse also does not respond to CDNs in vitro and in vivo. Lastly, Pneumovax 23, with an efficacy that depends on TMEM173, is less effective in mHAQ mice than in wild type mice. We conclude that HAQ is a null TMEM173 allele. Our findings have a significant impact on research related to MPYS-mediated human diseases and medicine.

The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons.

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.

The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo.

Effective mucosal adjuvants enhance the magnitude and quality of the vaccine response. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant. However, its in vivo mechanisms are unclear. Here, we showed, in mice, that CDG elicits stronger Ab and TH responses than the mammalian 2'3'-cyclic GMP-AMP (cGAMP), and generated better protection against Streptococcus pneumoniae infection than 2'3'-cGAMP adjuvanted vaccine. We identified two in vivo mechanisms of CDG. First, intranasally administered CDG greatly enhances Ag uptake, including pinocytosis and receptor-mediated endocytosis in vivo. The enhancement depends on MPYS (STING, MITA) expression in CD11C(+) cells. Second, we found that CDG selectively activated pinocytosis-efficient-DCs, leading to T(H) polarizing cytokines IL-12p70, IFNγ, IL-5, IL-13, IL-23, and IL-6 production in vivo. Notably, CDG induces IFNλ, but not IFNß, in vivo. Our study revealed previously unrecognized in vivo functions of MPYS and advanced our understanding of CDG as a mucosal vaccine adjuvant.

Confirmation of psoriasis susceptibility loci on chromosome 6p21 and 20p13 in French families.

Plaque psoriasis is a chronic inflammatory disorder of the skin. It is inherited as a multifactorial trait, with a strong genetic component. Linkage studies have identified a large number of disease loci, but very few could be replicated in independent family sets. In this study, we present the results of a genome-wide scan carried out in 14 French extended families. Candidate regions were then tested in a second set of 32 families. Analysis of the pooled samples confirmed linkage to chromosomes 6p21 (Z(MLB) score=3.5, P=0.0002) and 20p13 (Z(MLB) score=2.9, P=0.002), although there was little contribution of the second family set to the 20p13 linkage signal. Moreover, we identified four additional loci potentially linked to psoriasis. The major histocompatibility complex region on 6p21 is a major susceptibility locus, referred to as PSORS1, which has been found in most of the studies published to date. The 20p13 locus segregates independently of PSORS1 in psoriasis families. It has previously been thought to be involved in the predisposition to psoriasis and other inflammatory disorders such as atopic dermatitis (AD) and asthma. Although psoriasis and AD rarely occur together, this reinforces the hypothesis that psoriasis is influenced by genes with general effects on inflammation and immunity.

Prospective evaluation of risk factors of cutaneous drug reactions to sulfonamides in patients with AIDS.

BACKGROUND: Persons with HIV infection have increased rates of drug eruptions. OBJECTIVE: Our aim was to evaluate the risk factors of drug eruptions in response to sulfonamides in patients with AIDS, using a case-control analysis. METHODS: One hundred thirty-six patients who were hospitalized for pneumocystosis or toxoplasmosis were evaluated at the onset of treatment for various risk factors, which were then compared among patients with (48, 36%) and without (88, 64%) a drug eruption. RESULTS: In multivariate analysis, high CD8(+) cell count and age less than 36 years indicated a risk of drug eruption (respective odds ratios: 3.5 [95% CI 1.6-7.8], P =.002, and 2.1 [95% CI 1-4.6], P =.06). Markers of viral replication for HIV, Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, and parvovirus B19, slow acetylation phenotype or genotype, and glutathione level were not associated with a risk. Administration of corticosteroids had no preventive effect. CONCLUSIONS: Our results challenge several current concepts regarding drug eruptions by discarding a strong association with glutathione deficiency, slow acetylation, or active viral infections and by showing no preventive effect of corticosteroids.

Physicians' response to a letter to confirm diagnosis in a genetic study of psoriasis.

Context. Phenotyping of diseases is essential for genetic studies. In common diseases like psoriasis, the co-operation of family practitioners is helpful to confirm the phenotype of the patients. A genetic study was initiated in France by Généthon in 1996 in order to localise and identify genes for susceptibility to psoriasis. We used an unusual means to confirm psoriasis diagnosis: letters sent to patients' practitioners. Objective. To evaluate the efficiency of this means, and analyse the factors influencing the response rate of patients' physicians to a letter designed to confirm a diagnosis of psoriasis. Design. In the cases of putative psoriasis diagnosis (evaluated by systematic phone calls to the patients by a dermatologist), and with the agreement of the patients, a letter was sent to the patient's referent physician. We evaluated response rate to the letter. Results. 533 letters concerning 762 patients were sent to 456 physicians. The global response rate was 56.5%. The characteristics which modified the response rate were: the quality of the letter sent and some physicians' characteristics: dermatologists versus GPs, hospital versus private practice, and size of the town in which the physician had his practice. Conclusions. The use of letters to physicians as a means of phenotyping patients' diseases is an efficient method of confirming clinical diagnosis and should be used more frequently to confirm clinical diagnosis in large genetic studies. Some parameters such as ease of reply to the letter, whether the physician was a GP or a specialist, and the type of practice increased the response rate.