Interferon regulatory factor 8-deficiency determines massive neutrophil recruitment but T cell defect in fast growing granulomas during tuberculosis

Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8−/−) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8−/− mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8−/−, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8−/− mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions

Potent In Vitro Activity of Citrus aurantium Essential Oil and Vitis vinifera Hydrolate Against Gut Yeast Isolates from Irritable Bowel Syndrome Patients\u2014The Right Mix for Potential Therapeutic Use

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Proinflammatory and Cancer-Promoting Pathobiont Fusobacterium nucleatum Directly Targets Colorectal Cancer Stem Cells

Intestinal bacterial communities participate in gut homeostasis and are recognized as crucial in bowel inflammation and colorectal cancer (CRC). Fusobacterium nucleatum (Fn), a pathobiont of the oral microflora, has recently emerged as a CRC-associated microbe linked to disease progression, metastasis, and a poor clinical outcome; however, the primary cellular and/or microenvironmental targets of this agent remain elusive. We report here that Fn directly targets putative colorectal cancer stem cells (CR-CSCs), a tumor cell subset endowed with cancer re-initiating capacity after surgery and chemotherapy. A patient-derived CSC line, highly enriched (70%) for the stem marker CD133, was expanded as tumor spheroids, dissociated, and exposed in vitro to varying amounts (range 100-500 MOI) of Fn. We found that Fn stably adheres to CSCs, likely by multiple interactions involving the tumor-associated Gal-GalNac disaccharide and the Fn-docking protein CEA-family cell adhesion molecule 1 (CEACAM-1), robustly expressed on CSCs. Importantly, Fn elicited innate immune responses in CSCs and triggered a growth factor-like, protein tyrosine phosphorylation cascade largely dependent on CEACAM-1 and culminating in the activation of p42/44 MAP kinase. Thus, the direct stimulation of CSCs by Fn may contribute to microbiota-driven colorectal carcinogenesis and represent a target for innovative therapies

Haemophilus parasuis (Glaesserella parasuis) as a Potential Driver of Molecular Mimicry and Inflammation in Rheumatoid Arthritis

Background: Haemophilus parasuis (Hps; now Glaesserella parasuis) is an infectious agent that causes severe arthritis in swines and shares sequence similarity with residues 261–273 of collagen type 2 (Coll261−273), a possible autoantigen in rheumatoid arthritis (RA). Objectives/methods: We tested the presence of Hps sequencing 16S ribosomal RNA in crevicular fluid, synovial fluids, and tissues in patients with arthritis (RA and other peripheral arthritides) and in healthy controls. Moreover, we examined the cross-recognition of Hps by Coll261−273-specific T cells in HLA-DRB1*04pos RA patients, by T-cell receptor (TCR) beta chain spectratyping and T-cell phenotyping. Results: Hps DNA was present in 57.4% of the tooth crevicular fluids of RA patients and in 31.6% of controls. Anti-Hps IgM and IgG titers were detectable and correlated with disease duration and the age of the patients. Peripheral blood mononuclear cells (PBMCs) were stimulated with Hps virulence-associated trimeric autotransporter peptide (VtaA10755−766), homologous to human Coll261−273 or co-cultured with live Hps. In both conditions, the expanded TCR repertoire overlapped with Coll261−273 and led to the production of IL-17. Discussion: We show that the DNA of an infectious agent (Hps), not previously described as pathogen in humans, is present in most patients with RA and that an Hps peptide is able to activate T cells specific for Coll261−273, likely inducing or maintaining a molecular mimicry mechanism. Conclusion: The cross-reactivity between VtaA10755−766 of a non-human infectious agent and human Coll261−273 suggests an involvement in the pathogenesis of RA. This mechanism appears emphasized in predisposed individuals, such as patients with shared epitope

Monosodium urate crystals promote innate anti-mycobacterial immunity and improve BCG efficacy as a vaccine against tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination

Impact of pe_pgrs33 gene polymorphisms on mycobacterium tuberculosis infection and pathogenesis

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (Mtb\uce\u9433) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the Mtb\uce\u9433 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, Mtb\uce\u9433 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of Mtb\uce\u9433 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb

A rapid diagnostic workflow for cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae detection from blood cultures by MALDI-TOF mass spectrometry

Background: Nowadays, the global spread of resistance to oxyimino-cephalosporins in Enterobacteriaceae implies the need for novel diagnostics that can rapidly target resistant organisms from these bacterial species. Methods: In this study, we developed and evaluated a Direct Mass Spectrometry assay for Beta-Lactamase (D-MSBL) that allows direct identification of (oxyimino)cephalosporin-resistant Escherichia coli or Klebsiella pneumoniae from positive blood cultures (BCs), by using the matrix-assisted laser desorption ionization\ue2\u80\u93time of flight mass spectrometry (MALDI-TOF MS) technology. Results: The D-MSBL assay was performed on 93 E. coli or K. pneumoniae growing BC samples that were shortly co-incubated with cefotaxime (CTX) as the indicator cephalosporin. Susceptibility and resistance defining peaks from the samples\ue2\u80\u99 mass spectra were analyzed by a novel algorithm for bacterial organism classification. The D-MSBL assay allowed discrimination between E. coli and K. pneumoniae that were resistant or susceptible to CTX with a sensitivity of 86.8% and a specificity of 98.2%. Conclusion: The proposed algorithm-based D-MSBL assay, if integrated in the routine laboratory diagnostic workflow, may be useful to enhance the establishment of appropriate antibiotic therapy and to control the threat of oxyimino-cephalosporin resistance in hospital

The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies

Evaluation of PE_PGRS33 as a potential surface target for humoral responses against Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) PE_PGRS33 is a surface-exposed protein that was shown to interact with Toll-like receptor 2 on host macrophages to induce inflammatory signals and promote entry in macrophages. In this study, we investigated PE_PGRS33 as a potential target of a humoral response aimed at hampering key processes in tuberculosis pathogenesis. PE_PGRS33 protein was successfully expressed and purified under native condition in Escherichia coli. The purified protein retained its native functional and biological properties, showing the ability to elicit proinflammatory signals in murine and human macrophages. Interestingly, a polyclonal antiserum raised against native PE_PGRS33 showed no cross-reactions with other mycobacterial proteins. The anti-PE_PGRS33 serum was also able to inhibit Mtb entry into macrophages, but it did not reduce entry of the Mtb\u394pe_pgrs33 strain. Addition of native recombinant PE_PGRS33 to the Mtb\u394pe_pgrs33 strain during infection restored the Mtb wild-type entry phenotype in macrophage. Moreover, the anti-PE_PGRS33 serum was able to neutralize the proinflammatory activity of PE_PGRS33 in vitro. Furthermore, mice immunized with native recombinant PE_PGRS33, but not with a DNA vaccine expressing PE_PGRS33, were able to restrict M. smegmatis in vivo. These results highlight the potential use of PE_PGRS33 as a target of a neutralizing humoral response against tuberculosis