HMGN2 regulates non-tuberculous mycobacteria survival via modulation of M1 macrophage polarization.

Non-tuberculous mycobacteria (NTM), also known as an environmental and atypical mycobacteria, can cause the chronic pulmonary infectious diseases. Macrophages have been suggested as the main host cell to initiate the innate immune responses to NTM infection. However, the molecular mechanism to regulate the antimicrobial immune responses to NTM is still largely unknown. Current study showed that the NTM clinical groups, Mycobacterium abscessus and Mycobacterium smegmatis, significantly induced the M1 macrophage polarization with the characteristic production of nitric oxide (NO) and marker gene expression of iNOS, IFNγ, TNF-α, IL1-ß and IL-6. Interestingly, a non-histone nuclear protein, HMGN2 (high-mobility group N2), was found to be spontaneously induced during NTM-activated M1 macrophage polarization. Functional studies revealed that HMGN2 deficiency in NTM-infected macrophage promotes the expression of M1 markers and the production of NO via the enhanced activation of NF-κB and MAPK signalling. Further studies exhibited that HMGN2 knock-down also enhanced IFNγ-induced M1 macrophage polarization. Finally, we observed that silencing HMGN2 affected the survival of NTM in macrophage, which might largely relevant to enhanced macrophage polarization into M1 phenotype under the NTM infection. Collectively, current studies thus suggested a novel function of HMGN2 in regulating the anti-non-tuberculous mycobacteria innate immunity of macrophage.

Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8-cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and ß), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1ß secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

Mycobacterium smegmatis in skin biopsy specimens from patients with suppurative granulomatous inflammation.

Formalin-fixed, paraffin-embedded skin biopsy specimens, including 72 suppurative granulomatous inflammation (SGI) and 47 non-SGI controls, were tested for mycobacteria by using a broad-range PCR and a suspension array identification system. Mycobacterium smegmatis was detected in 13 (18.1%) of the SGI skin biopsy specimens, which was significantly more than 2 (4.3%) in the controls (odds ratio, 5.73; 95% confidence interval, 1.21 to 27.06; P = 0.028).

Rapid identification and detection of intracellular survival testing of mycobacterium smegmatis mc²155 that contains eis gene from mycobacterium tuberculosis by flow cytometry.

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. The enhanced intracellular survival (eis) gene (Rv2416c) from M. tuberculosis has been identified as a potential factor that can enhance the intracellular survival of Mycobacterium smegmatis in the macrophage cell line. However, the time requirements for intracellular survival testing of Mycobacterium using classical methodologies are still too long. In this study, we used M. smegmatis mc²155 that contains eis to develop and study a rapid method to test intracellular survival using flow cytometry. We demonstrated the success of this technique, which required only a few hours. This assay is rapid, accurate, and reproducible, and it would be valuable for the rapid detection of intracellular survival of mycobacteria.

Characterization of a novel rapidly growing Mycobacterium species associated with sepsis.

A rapidly growing mycobacterium was isolated five times from blood cultures from a 6-year-old female patient with relapsed pre-B-cell acute lymphocytic leukemia. All five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA gene, indicative of a single species. High-performance liquid chromatography analysis of mycolic acids indicated that the species was similar to Mycobacterium smegmatis. Sequence analysis of the 16S rRNA gene (1,455 bp) for one isolate demonstrated that the species was closely related to Mycobacterium diernhoferi. Based on the phenotypic features and phylogenetic analysis, it was concluded that the isolates represented a novel rapidly growing Mycobacterium species. The name "Mycobacterium hackensackense" is proposed for this unique strain, 147-0552(T), which was deposited in the American Type Culture Collection as ATCC BAA-823(T).

Panniculitis, due to Mycobacterium smegmatis, in two Finnish cats.

Pyogranulomatous panniculitis due to infection by Mycobacterium smegmatis was diagnosed in two cats in Finland, a country with a rather cold climate. The diagnosis was confirmed by sequencing of the 16S rRNA gene, which gave a perfect match with the M smegmatis strain ATCC 19420. Gene sequencing makes it possible to distinguish M smegmatis from closely related mycobacteria such as M goodii sp.nov. Diagnosing this entity seems to be a question of having a high index of suspicion. The appearance of the disease as well as sampling is described in detail. In our first case an initial erroneous diagnosis of Nocardia species considerably delayed our arriving at the right diagnosis. The first patient has now been followed for more than 7 years. Her disease is chronic, but she is not systemically affected. Several antimicrobials were tried. Probable side effects of enrofloxacin medication are described.

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection.

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.

[Non-tubercular mycobacterial infection of the lungs due to Mycobacterium smegmatis]. / Nichttuberkulöse Mykobakteriose der Lungen durch Mycobacterium smegmatis.

Nontuberculous mycobacteriosis due to M. smegmatis is a rarity. We report on the case of a 51 year old male HIV-seronegative patient without predisposing bronchopulmonary disease, but with a state after gastrectomy and splenectomy who developed unproductive cough, night sweat and weight loss. The chest radiograph and thoracic CT showed wide-spread bilateral patchy infiltrations. Histological examination of transbronchial biopsies revealed chronic carnificating pneumonia. A perhoracic fine-needle biopsy showed caseating epitheloid cell granulomas with acid fast bacilli. These were identified as M. smegmatis by PCR with subsequent sequencing. Acid fast bacilli could not be detected microscopically neither in sputum nor in bronchial secretions, however M. smegmatis has been repeatedly detected by culture in these materials. In neither material tubercle bacilli have been detected by nucleic acid amplification (NAT) or culture. Immunologic investigations revealed a reduced number of CD4+ lymphocytes and a reduction of interferon alpha- and -gamma-synthesis by peripheral blood mononuclear cells. Treatment with Rifabutin, Ethambutol, Clarithromycin and Ofloxacin resulted in complete clinical and roentgenological resolution.

Improved methods for immunoassay of mycothiol.

Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(N-maleimidopropionyl)biocytin. The MSH-biotin adduct was then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little as 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacterium spp. are described. The MSH-capture ELISA was tested for the detection of M. avium seeded in human urine or cerebrospinal fluid samples and for screening mutant M. smegmatis strains to detect MSH production.

Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus.

Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc(2)155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc(2)155, respectively.