Depletion of PD-1 or PD-L1 did not affect the mortality of mice infected with Mycobacterium avium.

The programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) pathway could affect antimicrobial immune responses by suppressing T cell activity. Several recent studies demonstrated that blocking of the PD-1/PD-L1 pathway exacerbated Mycobacterium tuberculosis infection. However, the effect of blocking this pathway in pulmonary Mycobacterium avium-intracellulare complex (MAC) infection is not fully understood. Wild-type, PD-1-deficient mice, and PD-L1-deficient mice were intranasally infected with Mycobacterium avium bacteria. Depletion of PD-1 or PD-L1 did not affect mortality and bacterial burden in MAC-infected mice. However, marked infiltration of CD8-positive T lymphocytes was observed in the lungs of PD-1 and PD-L1-deficient mice compared to wild-type mice. Comprehensive transcriptome analysis showed that levels of gene expressions related to Th1 immunity did not differ according to the genotypes. However, genes related to the activity of CD8-positive T cells and related chemokine activity were upregulated in the infected lungs of PD-1 and PD-L1-deficient mice. Thus, the lack of change in susceptibility to MAC infection in PD-1 and PD-L1-deficient mice might be explained by the absence of obvious changes in the Th1 immune response. Furthermore, activated CD8-positive cells in response to MAC infection in these mice seemed to not be relevant in the control of MAC infection.

Is vaccination a viable method to control Johne's disease caused by Mycobacterium avium subsp. paratuberculosis? Data from 12 million ovine vaccinations and 7.6 million carcass examinations in New South Wales, Australia from 1999-2009.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne's disease is increasing worldwide. In an attempt to control an epidemic of ovine Johne's disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naïve sheep population. METHODS: NSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair®) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology. PRINCIPAL FINDINGS: From 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009, the target commonly espoused for an effective control program based on vaccination. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks. SIGNIFICANCE: We show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of ≤ 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions.

Novel recombinant Mce-truncated protein based ELISA for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in domestic livestock.

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.

Host cytosolic RNA sensing pathway promotes T Lymphocyte-mediated mycobacterial killing in macrophages.

Mycobacterial infection leads to activation of the RIG-I/MAVS/TBK1 RNA sensing pathway in macrophages but the consequences of this activation remains poorly defined. In this study, we determined that activation of this RNA sensing pathway stimulates ICAM-1 expression in M.avium-infected macrophage through the inhibition of the E3 ubiquitin ligase CRL4COP1/DET1. CRL4 when active targets the transcription factor ETV5 for degradation by the ubiquitin-proteasome system. In the absence of the ETV5 transcription factor, ICAM-1 expression is significantly decreased. The M.avium-induced ICAM-1 production is required for the formation of immune synapse between infected macrophages and antigen-specific CD4+ T lymphocytes, and is essential for CD4+ T lymphocyte-mediated mycobacterial killing in vitro and in mice. This study demonstrates a previously undefined mechanism by which a host cytosolic RNA sensing pathway contributes to the interplay between mycobacteria infected macrophages and antigen-specific T lymphocytes.

Mutation on lysX from Mycobacterium avium hominissuis impacts the host-pathogen interaction and virulence phenotype.

The lysX gene from Mycobacterium avium hominissuis (MAH) is not only involved in cationic antimicrobial resistance but also regulates metabolic activity. An MAH lysX deficient mutant was shown to exhibit a metabolic shift at the extracellular state preadapting the bacteria to the conditions inside host-cells. It further showed stronger growth in human monocytes. In the present study, the LysX activity on host-pathogen interactions were analyzed. The lysX mutant from MAH proved to be more sensitive toward host-mediated stresses such as reactive oxygen species. Further, the lysX mutant exhibited increased inflammatory response in PBMC and multinucleated giant cell (MGC) formation in human macrophages during infection studies. Coincidentally, the lysX mutant strain revealed to be more reproductive in the Galleria mellonella infection model. Together, these data demonstrate that LysX plays a role in regulating the bacillary load in host organisms and the lack of lysX gene facilitates MAH adaptation to intracellular host-habitat, thereby suggesting an essential role of LysX in the modulation of host-pathogen interaction.

TNF-Mediated Compensatory Immunity to Mycobacterium avium in the Absence of Macrophage Activation by IFN-γ.

Granuloma formation is a hallmark of several infectious diseases, including those caused by Mycobacterium sp These structures are composed of accumulations of inflammatory cells, and it has been shown that cytokines such as IFN-γ and TNF-α are required for granuloma assembly during M. avium infections in mice. Macrophages (MΦs) insensitive to IFN-γ (MIIG) mice have MΦs, monocytes, and dendritic cells that are unresponsive to IFN-γ. We observed that although IFN-γ-/- mice present an exacerbated infection, the same is not true for MIIG animals, where the same levels of protection as the wild-type animals were observed in the liver and partial protection in the spleen. Unlike IFN-γ-/- mice, MIIG mice still develop well-defined granulomas, suggesting that IFN-γ-mediated MΦ activation is not required for granuloma assembly. This work also shows that MIIG animals exhibit increased cell recruitment with higher CD4+ T cells numbers as well as increased IFN-γ and TNF-α expression, suggesting that TNF-α may have a role in protection and may compensate the lack of MΦ response to IFN-γ in the MIIG model. TNF-α-deficient MIIG mice (MIIG.TNF-α-/-) exhibited increased bacterial burdens when compared with MIIG mice. These results suggest that in the absence of IFN-γ signaling in MΦs, TNF-α has a protective role against M. avium.

Reciprocal control of Mycobacterium avium and Mycobacterium tuberculosis infections by the alleles of the classic Class II H2-Aß gene in mice.

Genetic control of host susceptibility to M. avium, an important lung pathogen of immune-compromised individuals, remains incompletely defined. Apart from the slc11a1 (Nramp1) gene, which plays a pivotal role in genetic control of a few intracellular pathogens, including M. avium, in mice, we know nothing about genetic loci determining susceptibility to and/or severity of M. avium-triggered disease. Previously, our lab developed a panel of H2-congenic, recombinant mouse strains for identification of the MHC genes involved in the control of M. tuberculosis infection. In the present study, we applied a few recombinant strains from this panel to study $ possible influence of allelic variations in classical Class II genes on the development of M. avium infection. Our results demonstrate a clear difference in lung pathology, post-infection survival time, lung neutrophil influx and corresponding chemokine/cytokine responses, as well as the degree of lung T lymphocyte activation, between mouse strains differing by the alleles of a single highly polymorphic Class II H2-Aß gene. Paradoxically, mice carrying the H2-Aßb allele, which provides a notable protective effect against M. tuberculosis compared to the H2-Aßj allele, were more susceptible to M. avium infection as indicated by several parameters of the disease. We discuss possible reasons for such a reciprocal expression of phenotypes determined by a single allelic variant during two "similar" infections that may concern differences in virulence, NO-sensitivity, intracellular life style and antigenic composition between these two mycobacterial species.

BCG Vaccination Induces M. avium and M. abscessus Cross-Protective Immunity.

Pulmonary non-tuberculous mycobacterial (NTM) infections particularly caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are becoming major health problems in the U.S. New therapies or vaccines which will help prevent the disease, shorten treatment duration and/or increase treatment success rates are urgently needed. This study was conducted with the objective of testing the hypothesis that Bacillus Calmette Guerin (BCG), a vaccine used for prevention of serious forms of tuberculosis (TB) in children and adolescents in tuberculosis hyperendemic countries, induces cross-protective T cell immunity against Mycobacterium avium (MAV) and MAB. Human TB and NTM cross-protective T cells were quantified using flow cytometric assays. The ability of BCG expanded T cells to inhibit the intracellular growth of MAV and MAB was assessed in co-cultures with infected autologous macrophages. In both BCG-vaccinated and M. tuberculosis (Mtb)-infected mice, NTM cross-reactive immunity was measured using IFN-γ ELISPOT assays. Our results demonstrate the following key findings: (i) peripheral blood mononuclear cells from TB skin test-positive individuals contain MAV and MAB cross-reactive T cells, (ii) both BCG vaccination and Mtb infection of mice induce MAV and MAB cross-reactive splenic cells, (iii) BCG-expanded T cells inhibit intracellular MAV and MAB, (iv) CD4, CD8, and γδ T cells play important roles in inhibition of intracellular MAV and MAB and (v) BCG vaccination of healthy volunteers induces TB and NTM cross-reactive T cells. In conclusion, BCG-vaccination induces NTM cross-reactive immunity, and has the potential for use as a vaccine or immunotherapy to prevent and/or treat pulmonary NTM disease.

Presence of Infected Gr-1intCD11bhiCD11cint Monocytic Myeloid Derived Suppressor Cells Subverts T Cell Response and Is Associated With Impaired Dendritic Cell Function in Mycobacterium avium-Infected Mice.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

Mycobacterium avium lysate induces matrix metalloproteinase-1 in intestinal tissue and peripheral blood: Observations from selected hospital based Zambian adults.

OBJECTIVES: Environmental enteropathy is prevalent in low-income countries, although its aetiology is unknown. We investigated if Mycobacterium avium antigens, which are commonly found in the environment, could contribute to its pathogenesis in a population known to have widespread environmental enteropathy. METHODS: Routine endoscopy patients at the University Teaching Hospital, Lusaka whose endoscopy results were normal submitted duodenal biopsies and whole blood samples. Samples were stimulated with M. avium lysate over 24h while unstimulated samples served as negative controls. Matrix metalloproteinase (MMP) and cytokine response in supernatants were quantified using ELISA and cytometric bead array. RESULTS: Samples from 48 patients (56% women) were analysed, with a median age of 35 years (IQR 27.5, 50.5). M. avium induced the secretion of a wide-range of Th1, Th2 and Th17 cytokines in blood but only IL-1ß and IL-6 in duodenal tissue. However it differentially induced the secretion of MMP-1 in duodenal tissue compared to negative controls (p=0.004). A similar MMP-1 response but with lower concentrations was observed in blood. CONCLUSION: The induction of MMP-1 and cytokines by M. avium in duodenal tissue suggests that environmental mycobacteria could contribute to the epithelial disruption seen in environmental enteropathy, and a need to further explore possible biomarkers that may predict this exposure in at-risk populations.

Ag85-focused T-cell immune response controls Mycobacterium avium chronic infection.

CD4+ T cells are essential players for the control of mycobacterial infections. Several mycobacterial antigens have been identified for eliciting a relevant CD4+ T cell mediated-immune response, and numerous studies explored this issue in the context of Mycobacterium tuberculosis infection. Antigen 85 (Ag85), a highly conserved protein across Mycobacterium species, is secreted at the early phase of M. tuberculosis infection leading to the proliferation of Ag85-specific CD4+ T cells. However, in the context of Mycobacterium avium infection, little is known about the expression of this antigen and the elicited immune response. In the current work, we investigated if a T cell receptor (TCR) repertoire mostly, but not exclusively, directed at Ag85 is sufficient to mount a protective immune response against M. avium. We show that P25 mice, whose majority of T cells express a transgenic TCR specific for Ag85, control M. avium infection at the same level as wild type (WT) mice up to 20 weeks post-infection (wpi). During M. avium infection, Ag85 antigen is easily detected in the liver of 20 wpi mice by immunohistochemistry. In spite of the propensity of P25 CD4+ T cells to produce higher amounts of interferon-gamma (IFNγ) upon ex vivo stimulation, no differences in serum IFNγ levels are detected in P25 compared to WT mice, nor enhanced immunopathology is detected in P25 mice. These results indicate that a T cell response dominated by Ag85-specific T cells is appropriate to control M. avium infection with no signs of immunopathology.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis.

Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Innate IFN-γ-Producing Cells Developing in the Absence of IL-2 Receptor Common γ-Chain.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.

Altered humoral immunity to mycobacterial antigens in Japanese patients affected by inflammatory demyelinating diseases of the central nervous system.

Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis (BCG) have been associated to several human autoimmune diseases such as multiple sclerosis (MS), but there are conflicting evidence on the issue. The objective of this study is to evaluate their role in Japanese patients affected by inflammatory demyelinating disorders of the central nervous system (IDDs). A total of 97 IDDs subjects including 51 MS and 46 neuromyelitis optica spectrum disorder (NMOSD) patients, and 34 healthy controls (HCs) were tested for the detection of IgG, IgM and IgA against mycobacterial antigens by indirect ELISA. The levels of anti-MAP IgG were higher in MS patients compared to NMOSD patients (AUC = 0.59, p = 0.02) and HCs (AUC = 0.67, p = 0.01), and the anti-MAP antibodies were more prevalent in MS patients treated with interferon-beta (OR = 11.9; p = 0.004). Anti-BCG IgG antibodies were detected in 8% of MS, 32% of NMOSD and 18% of HCs, the difference between MS and NMOSD groups was statistically significant (AUC = 0.66, p = 0.005). Competition experiments showed that nonspecific IgM were elicited by common mycobacterial antigens. Our study provided further evidence for a possible association between MAP and MS, while BCG vaccination seemed to be inversely related to the risk of developing MS.

Virulence and immunogenicity of genetically defined human and porcine isolates of M. avium subsp. hominissuis in an experimental mouse infection.

Mycobacterium avium subsp. hominissuis (Mah) represents a health concern for humans and to a lesser extent for pigs, but its zoonotic potential remains elusive. Using multispacer sequence typing (MST) we previously identified 49 different genotypes of Mah among Belgian clinical and porcine isolates, with 5 MSTs shared by both hosts. Using experimental intranasal infection of BALB/c mice, we compared the virulence and immunogenicity of porcine and clinical human isolates with shared genotype or with a genotype only found in humans or pigs. Bacterial replication was monitored for 20 weeks in lungs, spleen and liver and mycobacteria specific spleen cell IFN-γ, IL-10 and IL-17 production as well as serum antibody responses were analyzed. Isolates varied in virulence, with human and porcine isolates sharing MST22 genotype showing a thousand fold higher bacterial replication in lungs and more dissemination to spleen and liver than the human and porcine MST91 isolates. Virulent MST22 type was also associated with progressive suppression of IFN-γ and IL-17 responses, and increased IL-10 production. Whole genome sequencing of the two virulent isolates with MST22 genotype and two avirulent isolates of genotype MST91 and comparison with two well-studied M. avium subsp. hominissuis reference strains i.e. Mah 104 and Mah TH135, identified in the two MST22 isolates nine specific virulence factors of the mammalian cell entry family, that were identical with Mah 104 strain. Despite the obvious limitations of the mouse model, a striking link of virulence and identity at the genome level of porcine and human isolates with the same multisequence type, for which no correlation of place of residence (humans) or farm of origin (pigs) was observed, seems to point to the existence in the environment of certain genotypes of Mah which may be more infectious both for humans and pigs than other genotypes.

Combining HLA-DRB1-DQB1 and Mycobacterium Avium Subspecies Paratubercolosis (MAP) antibodies in Sardinian multiple sclerosis patients: associated or independent risk factors?

BACKGROUND: Amongst Sardinians the human leukocyte antigen (HLA) DRB1-DQB1 haplotypes *15:02-*06:01, *16:01-*05:02, *14:01-4-*05:03 are protective for multiple sclerosis (MS), while *13:03-*03:01, *04:05-*03:01, *03:01-*02:01, *15:01-*06:02 and Mycobacterium avium subspecies paratubercolosis (MAP) are predisposing factors. We studied the correlation between MAP and HLA. METHODS: Five hundred thirty-one patients were searched for anti-MAP2694 antibodies, DRB1-DQB1 genotyping was performed. The haplotypes were classified as predisposing, neutral or protective. RESULTS: Anti-MAP2694 were found in 23 % of subjects carrying one protective HLA versus 32 % without (p = 0.04). CONCLUSIONS: We showed a lower frequency of Abs in patients with protective HLA. These haplotypes could have a protective role for both MS and MAP.

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.

Soluble BAFF Level Is Not Correlated to Mycobacterium avium Subspecies Paratuberculosis Antibodies and Increases After Interferon-ß Therapy in Multiple Sclerosis Patients.

B cells are being recognized as one of the major players in the pathogenesis of multiple sclerosis (MS). The B cell activating factor (BAFF) system plays an essential role in B cell homeostasis and function in the periphery. Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to MS in Sardinia. Antibodies against a MAP surface protein, MAP_2694, have been found significantly associated to MS patients, and this response was modified by interferon-ß therapy. Increased BAFF levels following IFN-ß therapy have been also described in MS patients. In this study, we evaluated whether soluble BAFF levels are comparable in men and women affected by MS and performed a correlation of the reported BAFF increase in MS patients under IFN-ß therapy with changes of humoral response against MAP_2694. For these reasons, we investigated 44 MS patients before and after IFN-ß therapy. A significant difference of BAFF levels was found between men and women with MS; moreover, we confirmed that IFN-ß therapy strongly induces BAFF serum levels, but this was not related to the modification of immunological response against MAP_2694. In conclusion, our study highlights that IFN-ß therapy induces the potent B cell survival factor BAFF without alterations of the humoral immune response against MAP.

TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells.

Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum.

Oral Tolerance to Environmental Mycobacteria Interferes with Intradermal, but Not Pulmonary, Immunization against Tuberculosis.

Bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB) and is administered in over 150 countries worldwide. Despite its widespread use, the vaccine has a variable protective efficacy of 0-80%, with the lowest efficacy rates in tropical regions where TB is most prevalent. This variability is partially due to ubiquitous environmental mycobacteria (EM) found in soil and water sources, with high EM prevalence coinciding with areas of poor vaccine efficacy. In an effort to elucidate the mechanisms underlying EM interference with BCG vaccine efficacy, we exposed mice chronically to Mycobacterium avium (M. avium), a specific EM, by two different routes, the oral and intradermal route, to mimic human exposure. After intradermal BCG immunization in mice exposed to oral M. avium, we saw a significant decrease in the pro-inflammatory cytokine IFN-γ, and an increase in T regulatory cells and the immunosuppressive cytokine IL-10 compared to naïve BCG-vaccinated animals. To circumvent the immunosuppressive effect of oral M. avium exposure, we vaccinated mice by the pulmonary route with BCG. Inhaled BCG immunization rescued IFN-γ levels and increased CD4 and CD8 T cell recruitment into airways in M. avium-presensitized mice. In contrast, intradermal BCG vaccination was ineffective at T cell recruitment into the airway. Pulmonary BCG vaccination proved protective against Mtb infection regardless of previous oral M. avium exposure, compared to intradermal BCG immunization. In conclusion, our data indicate that vaccination against TB by the pulmonary route increases BCG vaccine efficacy by avoiding the immunosuppressive interference generated by chronic oral exposure to EM. This has implications in TB-burdened countries where drug resistance is on the rise and health care options are limited due to economic considerations. A successful vaccine against TB is necessary in these areas as it is both effective and economical.