Progress of the Art of Macrophage Polarization and Different Subtypes in Mycobacterial Infection.

Mycobacteriosis, mostly resulting from Mycobacterium tuberculosis (MTb), nontuberculous mycobacteria (NTM), and Mycobacterium leprae (M. leprae), is the long-standing granulomatous disease that ravages several organs including skin, lung, and peripheral nerves, and it has a spectrum of clinical-pathologic features based on the interaction of bacilli and host immune response. Histiocytes in infectious granulomas mainly consist of infected and uninfected macrophages (Mφs), multinucleated giant cells (MGCs), epithelioid cells (ECs), and foam cells (FCs), which are commonly discovered in lesions in patients with mycobacteriosis. Granuloma Mφ polarization or reprogramming is the crucial appearance of the host immune response to pathogen aggression, which gets a command of endocellular microbe persistence. Herein, we recapitulate the current gaps and challenges during Mφ polarization and the different subpopulations of mycobacteriosis.

Flow Cytometry Analysis of Mycobacteria and Mycobacteria-Infected Immune Cells.

Flow cytometry enables the measurement of tens of features on individual cells from complex mixtures. Flow cytometry enables high-throughput quantification of cell size, gene and protein expression. In the case of studies of host-pathogen interactions, this tool provides a facile way of identifying cells that have been successfully infected by a pathogen. Several recent technological advances have greatly improved throughput and the number of features that can be simultaneously monitored by this technique. Here, we describe common workflows to study Mycobacterium tuberculosis heterogeneity and host-M. tuberculosis interactions using flow cytometry and related technologies.

Bcl2 negatively regulates Protective Immune Responses During Mycobacterial Infection.

We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-culturing human PBMCs and BMDCs with antigen-primed T cells increased their proliferation, activation and effector function. These results point towards a critical role for Bcl2 in regulating BMDCs defense responses to mycobacterial infection.

Histopathology of laboratory-reared Nothobranchius fishes: Mycobacterial infections versus neoplastic lesions.

Short-lived killifishes of the genus Nothobranchius Peters, 1868 (Cyprinodontiformes) are considered promising model organisms for biomedical research on ageing and tumorigenesis. We conducted histopathological analysis of 411 adult individuals from three Nothobranchius species to study details on spontaneous age-related neoplastic lesions. Light microscopy based on H&E and toluidine blue-stained sections revealed (a) non-proliferative liver changes with pronounced vacuolation of hepatocytes; (b) proliferation of kidney haemopoietic tissue contributing to excretory system damage; (c) proliferation of splenic mononuclear haemoblasts accompanied by reduced erythropoiesis; (d) proliferation of mononuclear cell aggregates in the liver parenchyma; and (e) rare occurrence of hepatocellular adenomas. Ziehl-Neelsen (ZN) staining revealed that the proliferative lesions are a host defence response to mycobacterial infections manifested by activation of the mononuclear phagocytic system and atypical granulomatous inflammatory reaction. 16S rRNA analysis identified three species of Mycobacterium in our samples. Our findings turn attention to lesions which mimic neoplasms by their gross appearance and question the light microscopic interpretation of lesions unless differential ZN staining is included. Beyond the limitations of our morphological approach, the intensity of mycobacterial infections is a challenging opportunity for research into the molecular-genetic background of the mononuclear phagocytic system reaction in Nothobranchius killifish.

A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea).

We detail a novel presentation of tuberculosis associated with intestinal perforation in an endangered Australian sea lion (Neophoca cinerea) from South Australian waters and confirm the presence of this disease in the region of highest pup production. In February 2017, a 3-yr-old juvenile male died shortly after hauling out at the Kingscote beach on Kangaroo Island. On postmortem examination, we found a mid-jejunal intestinal perforation and partial obstruction (from a strangulating fibrous and granulomatous mesenteric mass), a marked multicentric abdominal fibrosing granulomatous lymphadenitis, and a large volume serosanguinous peritoneal effusion. Acid-fast bacteria were detected postmortem in cytologic preparations of the mesenteric lymph node and in histologic sections of jejunum and the encircling mass. Mycobacterial infection was confirmed by positive culture after 3 wk. Molecular typing using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing with 12-locus analysis identified Mycobacterium pinnipedii. This case highlights the need for vigilance of zoonotic disease risk when handling pinnipeds, including in the absence of specific respiratory signs or grossly apparent pulmonary pathology. Increased serologic population surveillance is recommended to assess the species' risk from this and other endemic diseases, especially given its endangered status.

Caprine nodular thelitis due to Mycobacterium uberis: A series of 26 cases in 11 dairy goat farms in Western France.

Bovine Nodular Thelitis (BNT) is a granulomatous dermatitis of teat skin associated with acid-fast bacilli. A similar condition has been recorded in a dairy goat flock in France recently. The causative agent was shown to be related to the leprosy-causing bacilli Mycobacterium leprae and M. lepromatosis, then sequenced and named M. uberis. Following the initial report in goats, the aim of this study was to investigate new cases of Caprine Nodular Thelitis (CNT) in the same area to confirm the presence of M. uberis by molecular techniques and to get a better description of the clinical signs and of the affected flocks. Twenty-six animals (25 females and 1 male) from 11 flocks were included in the study. Lesions were located on the udder/teat skin (24/25), on the body skin (6/25) or on the scrotum skin (1/1). Udder skin lesions were circular, nodular and/or ulcerate covered with a crust and associated with supramammary lymph node enlargement. Body skin lesions were located at different parts of the body, showed large necrotizing ulcers with undetermined edges and were associated with regional lymph node enlargement. Histopathological results indicated granulomatous dermatitis and lymphadenitis of varying intensity with no acid-fast bacilli seen after Fite-Faraco staining. M. uberis DNA was amplified from 26 samples out of 47 (udder: 11/22; lymph node: 11/20; body: 4/5). The female goats were mostly older than 4 year of age and originated from breeding units characterized by large flock size and high proportion of goat in continuous lactation.

TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages.

The implications of TLR-2 mediated alterations in cytosolic-Ca2+((Ca2+)c) levels in M. smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus, we observed TLR-2 signalling is required in the phagocytosis of M. smegmatis. M. smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca2+)c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca2+)c-surge triggered downstream ER-stress and superoxide (O2-) generation. The cross-talk between ER-stress and O2- amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O2--NO axis imperative in M. smegmatis-induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca2+)c-surge and ensuing cross-talk between ER-stress and O2- potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.

Advances in Cardiovascular Disease Lipid Research Can Provide Novel Insights Into Mycobacterial Pathogenesis.

Cardiovascular disease (CVD) is the leading cause of death in industrialized nations and an emerging health problem in the developing world. Systemic inflammatory processes associated with alterations in lipid metabolism are a major contributing factor that mediates the development of CVDs, especially atherosclerosis. Therefore, the pathways promoting alterations in lipid metabolism and the interplay between varying cellular types, signaling agents, and effector molecules have been well-studied. Mycobacterial species are the causative agents of various infectious diseases in both humans and animals. Modulation of host lipid metabolism by mycobacteria plays a prominent role in its survival strategy within the host as well as in disease pathogenesis. However, there are still several knowledge gaps in the mechanistic understanding of how mycobacteria can alter host lipid metabolism. Considering the in-depth research available in the area of cardiovascular research, this review presents an overview of the parallel areas of research in host lipid-mediated immunological changes that might be extrapolated and explored to understand the underlying basis of mycobacterial pathogenesis.

Atypical bronchial carcinoid with postobstructive mycobacterial infection: case report and review of literature.

BACKGROUND: Pulmonary carcinoids are included in the group of neuroendocrine tumors (NET) and derive from pulmonary neuroendocrine cells. The incidence of these tumors is increasing, but disease awareness remains low among clinicians. The synchronous presentation of lung cancer and mycobacterial infection is well known but the combination of pulmonary carcinoid and mycobacterial infection is rare. CASE PRESENTATION: We treated a 45-year-old female who presented with recurrent pneumonia. Chest X-ray showed a consolidation in the left upper lobe. The patient was treated with various courses of antibiotics without full recovery after six months. Computed tomography (CT) scan demonstrated a central mass in the left upper lobe. Bronchoscopy revealed an endobronchial, well-defined lesion that totally obstructed the left upper lobe bronchus. Bronchial biopsy showed typical carcinoid tumor. Rigid bronchoscopy with electrocautery was attempted, but we were unable to radically remove the tumor. Therefore lobectomy was performed. The surgical pathology specimen showed atypical bronchial carcinoid and consolidations in the lung parenchyma with granulomatous inflammation distally of the bronchial obstruction. Ziehl-Neelsen staining demonstrated acid fast bacilli indicative of mycobacterial infection. CONCLUSIONS: This case history illustrates the importance of careful surgical pathologic examination, not only of the resected tumor, but also of the postobstructive lung parenchyma. Specific postobstructive infections such as tuberculosis or nontuberculous mycobacteria (NTM) can have clinical implications.

Role of CT-guided transthoracic biopsy in the diagnosis of mycobacterial infection.

Mycobacterial infection(MI) is sometimes diagnosed using CT-guided transthoracic needle biopsy (TNB). However, the exact role of CT-guided TNB in this diagnostic process is not clearly known. The purpose of this study is to analyze the role of CT-guided TNB in patients with MI who present with a focal lung lesion. Of 1233 patients who underwent CT-guided TNB from January 2010 through February 2016 at our institution, patients with a final diagnosis of MI were included for analysis. Clinical characteristics and biopsy-related factors were compared between patients whose diagnosis could be established using TNB samples alone (group 1) and those whose samples from additional tests were necessary for diagnosis (group 2). We also analyzed the possible benefit of CT-guided TNB as compared with bronchoscopy in a subgroup who underwent both procedures. 47 patients with MI were included in the study, with 37 patients (78.7%) in group 1 and 10 patients (21.2%) in group 2. There was no statistically significant difference in clinical characteristics or biopsy-related factors between the two groups. Of 41 patients with MI who underwent both bronchoscopy and TNB, success in diagnosis was solely attributable to TNB in 16 (39.0%) patients; in 19 (46.0%) patients, diagnosis could be made based on bronchoscopy results alone. MI can be successfully diagnosed by CT-guided TNB in about 80% of patients with MI who underwent TNB, but 46% of the patients could have been diagnosed with bronchoscopy results alone. CT-guided TNB is inferior to bronchoscopy in the differentiation of Mycobacterium species even in peripheral lung lesions.

MULTIMODAL IMAGING OF CHOROIDAL LESIONS IN DISSEMINATED MYCOBACTERIUM CHIMAERA INFECTION AFTER CARDIOTHORACIC SURGERY.

PURPOSE: To explore morphologic characteristics of choroidal lesions in patients with disseminated Mycobacterium chimaera infection subsequent to open-heart surgery. METHODS: Nine patients (18 eyes) with systemic M. chimaera infection were reviewed. Activity of choroidal lesions were evaluated using biomicroscopy, fundus autofluorescence, enhanced depth imaging optical coherence tomography, fluorescein angiography/indocyanine green angiography, and optical coherence tomography angiography. Relationships of choroidal findings to systemic disease activity were sought. RESULTS: All 9 male patients, aged between 49 and 66 years, were diagnosed with endocarditis and/or aortic graft infection. Mean follow-up was 17.6 months. Four patients had only inactive lesions (mild disease). In all five patients (10 eyes) with progressive ocular disease, indocyanine green angiography was superior to other tests for revealing new lesions and active lesions correlated with hyporeflective choroidal areas on enhanced depth imaging optical coherence tomography. One eye with a large choroidal granuloma developed choroidal neovascularization. Optical coherence tomography angiography showed areas with reduced perfusion at the inner choroid. All 5 patients with progressive ocular disease had evidence of systemic disease activity within ±6 weeks' duration. CONCLUSION: Choroidal manifestation of disseminated M. chimaera infection indicates systemic disease activity. Multimodal imaging is suitable to recognize progressive ocular disease. We propose ophthalmologic screening examinations for patients with M. chimaera infection.

Phospholipase C-γ2 promotes intracellular survival of mycobacteria.

Mycobacterium tuberculosis (Mtb) infects millions of people each year. These bacilli can survive inside macrophages. To favor their survival, pathogen alters various signal transduction pathways in host cells. Phospholipase C (PLC) signaling regulates various processes in mammalian cells but has never been investigated for their roles in regulating phagocytosis and killing of mycobacteria by macrophages. Here, we report that infection with Mtb but not Mycobacterium smegmatis (MS) induces phosphorylation of PLC-γ2 at tyrosine 1217 in J774A.1 cells. Small interfering RNA-mediated knockdown of PLC-γ2 expression leads to the enhanced killing of both MS and Mtb by these cells suggesting that Mtb activates PLC-γ2 to promote its intracellular survival within macrophages. Knockdown of PLC-γ2 also lead to increased uptake of Mtb but not MS by J774.A.1 cells. Further, we have observed that PLC-γ2 was required for Mtb-induced inhibition of expression of proinflammatory cytokine tumor necrosis factor-α, inducible nitric oxide synthase, and chemokine (C-C motif) ligand 5 (RANTES). Altogether, our results for the first time demonstrate that Mtb induces activation of macrophages PLC-γ2 to inhibit their mycobactericidal response.

Hemophagocytic syndrome in patients living with HIV: a retrospective study.

Various infectious diseases can hyper-stimulate the immune system, causing hemophagocytic syndrome (HPS). Little is known regarding the accuracy of diagnostic criteria and epidemiological triggering factors in the acquired immunodeficiency syndrome (AIDS) setting. We investigated the major infectious disease triggers of HPS in patients living with human immunodeficiency virus (HIV)/AIDS and determined the accuracy of bone marrow aspiration (BMA). The inclusion criteria were (i) confirmed HIV diagnosis, (ii) bone marrow aspiration, and (iii) a minimum of four HPS criteria. Patients were further classified into those with four presumed HPS criteria, or ≥ 5 confirmed criteria. The disease triggers, accuracy of bone marrow aspiration, and prognosis markers were examined. Presumed HPS was observed in 15/36 patients (41%), and confirmed HPS in 58% (n = 21). The major etiological triggers were infection with Mycobacterium (34%), Cytomegalovirus (14%), Cryptococcus neoformans (11%), and hematological or tumoral disease (11%). BMA demonstrated 93% specificity on screening diagnosis (odds ratio [OR] 12.7, 95% confidence interval [CI] 1.4-115.1, P = 0.01). Ferritin > 5000 ng/mL correlated with probability of death in univariate analysis (OR 6.00, 95% CI 1.33-27.05, P = 0.02). Ferritin performance as test of death probability presented area under the curve as 0.74 (95% CI 0.56-0.91, P = 0.016). However, neither cluster of differentiation for lymphocyte count nor HIV viral load correlated with patient deaths. Mycobacterium spp. and Cytomegalovirus were the main factors triggering HPS, followed by Cryptococcus neoformans, and hematological and tumoral diseases. High ferritin levels were associated with increased death probability. High specificity was noted with BMA.

Plasticity of antimicrobial and phagocytic programs in human macrophages.

Macrophage (MΦ) polarization is triggered during the innate immune response to defend against microbial pathogens, but can also contribute to disease pathogenesis. In a previous study, we found that interleukin-15 (IL-15) -derived classically activated macrophages (M1 MΦ) have enhanced antimicrobial activity, whereas IL-10-derived alternatively activated macrophages (M2 MΦ) were highly phagocytic but lacked antimicrobial activity. Given that the ability to modulate MΦ polarization from M2 MΦ to M1 MΦ may promote a more effective immune response to infection, we investigated the plasticity of these MΦ programs. Addition of IL-10 to M1 MΦ induced M2-like MΦ, but IL-15 had little effect on M2 MΦ. We determined the set of immune receptors that are present on M2 MΦ, elucidating two candidates for inducing plasticity of M2 MΦ, Toll-like receptor 1 (TLR1) and interferonγ (IFN-γ) receptor 1. Stimulation of M2 MΦ with TLR2/1 ligand (TLR2/1L) or IFN-γ alone was not sufficient to alter M2 MΦ phenotype or function. However, co-addition of TLR2/1L and IFN-γ re-educated M2 MΦ towards the M1 MΦ phenotype, with a decrease in the phagocytosis of lipids and mycobacteria, as well as recovery of the vitamin-D-dependent antimicrobial pathway compared with M2 MΦ maintained in polarizing conditions. Similarly, treatment of M2 MΦ with both TLR2/1L and anti-IL-10 neutralizing antibodies led to polarization to the M1-like MΦ phenotype and function. Together, our data demonstrate an approach to induce MΦ plasticity that provides the potential for re-educating MΦ function in human mycobacterial disease to promote host defense and limit pathogenesis.

Retrospective Evaluation of Clinical Signs and Gross Pathologic Findings in Birds Infected With Mycobacterium genavense.

Mycobacterium genavense is regarded as the primary cause of mycobacteriosis in passerine and psittacine birds kept in captivity. Mycobacterium genavense is a potential zoonotic pathogen; therefore, early antemortem detection in birds is needed. In humans, infections with M genavense are found predominantly in immunocompromised people. To investigate clinical signs and pathologic lesions and to determine the prevalence of coinfections in birds infected with M genavense, we reviewed records of 83 birds in which DNA from M genavense had been detected via real-time polymerase chain reaction. To evaluate clinical signs in birds presented as patients, results of standardized examinations of 60 birds and radiographic results from 37 birds were investigated. Necropsy results of 82 of the 83 birds were evaluated, including results of additional parasitologic, bacteriologic, and virologic examinations. Birds included in the study comprised 15 species in the orders Passeriformes, Psittaciformes, Coliiformes, Columbiformes, Coraciiformes, and Ciconiiformes. A wide range of clinical manifestations were documented, including neurologic disorders, ocular manifestations, and gastrointestinal signs. Of the 60 birds examined clinically, 15% showed no clinical signs. Coinfections with a wide range of pathogens were detected in 52% (43 of 83) of the tested birds. Coinfections included Macrorhabdus ornithogaster, circovirus, polyomavirus, avian bornavirus, adenovirus, Mycobacterium avium ssp. avium/ silvaticum, Mycoplasma species, Salmonella species, Escherichia coli, Aspergillus species, and various parasites. The high number of coinfections may reflect an impaired immune status in the birds examined. These results also suggest a broad host range for M genavense, and the existence of various clinical signs that may be strongly associated with coinfections with other pathogens.

[Feline leprosy in a 5-month-old male cat in Germany]. / Feline Lepra bei einem 5 Monate alten Kater in Deutschland.

A 5-month-old, male, 4 kg European shorthair cat presented with ulcera ted cutaneous nodules in many areas over its entire body. The serological results for feline immunodeficiency virus and the feline leukaemia virus were negative. Following detection of acid-proof bacilli in histological examination of three skin biopsies, the diagnosis of mycobacteriosis of the skin was made. Polymerase chain reaction revealed the presence of Mycobacteriumlepraemurium. After surgical excision of all cutaneous nodules and a 14.5-week antibiotic therapy with Rifampicin and Clarithromycin, the cat was classified as being cured. During the observation period of 1 year after the end of the therapy, no relapse occurred.