A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

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.

Functional Interplay between Type I and II Interferons Is Essential to Limit Influenza A Virus-Induced Tissue Inflammation.

Host control of influenza A virus (IAV) is associated with exuberant pulmonary inflammation characterized by the influx of myeloid cells and production of proinflammatory cytokines including interferons (IFNs). It is unclear, however, how the immune system clears the virus without causing lethal immunopathology. Here, we demonstrate that in addition to its known anti-viral activity, STAT1 signaling coordinates host inflammation during IAV infection in mice. This regulatory mechanism is dependent on both type I IFN and IFN-γ receptor signaling and, importantly, requires the functional interplay between the two pathways. The protective function of type I IFNs is associated with not only the recruitment of classical inflammatory Ly6Chi monocytes into IAV-infected lungs, but also the prevention of excessive monocyte activation by IFN-γ. Unexpectedly, type I IFNs preferentially regulate IFN-γ signaling in Ly6Clo rather than inflammatory Ly6Chi mononuclear cell populations. In the absence of type I IFN signaling, Ly6Clo monocytes/macrophages, become phenotypically and functionally more proinflammatory than Ly6Chi cells, revealing an unanticipated function of the Ly6Clo mononuclear cell subset in tissue inflammation. In addition, we show that type I IFNs employ distinct mechanisms to regulate monocyte and neutrophil trafficking. Type I IFN signaling is necessary, but not sufficient, for preventing neutrophil recruitment into the lungs of IAV-infected mice. Instead, the cooperation of type I IFNs and lymphocyte-produced IFN-γ is required to regulate the tissue neutrophilic response to IAV. Our study demonstrates that IFN interplay links innate and adaptive anti-viral immunity to orchestrate tissue inflammation and reveals an additional level of complexity for IFN-dependent regulatory mechanisms that function to prevent excessive immunopathology while preserving anti-microbial functions.

Epitope-specific CD4+, but not CD8+, T-cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection.

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4(+) and CD8(+) T-cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4(+) T-cell epitope from the Ag85B protein (PR8.p25) or CD8(+) T-cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T-cell responses to the M. tuberculosis-specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly-functional CD4(+) T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN-γ-producing M. tuberculosis-specific CD8(+) T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope-specific CD4(+), but not CD8(+) T cells, is essential for protection against acute M. tuberculosis infection in the lung.

Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

Molecular and cellular mechanisms of Mycobacterium avium-induced thymic atrophy.

Thymic atrophy has been described as a consequence of infection by several pathogens and shown to be induced through diverse mechanisms. Using the mouse model of Mycobacterium avium infection, we show in this study that the production of NO from IFN-γ-activated macrophages plays a major role in mycobacterial infection-induced thymic atrophy. Our results show that disseminated infection with a highly virulent strain of M. avium, but not with a low-virulence strain, led to a progressive thymic atrophy. Thymic involution was prevented in genetically manipulated mice unable to produce IFN-γ or the inducible NO synthase. In addition, mice with a selective impairment of IFN-γ signaling in macrophages were similarly protected from infection-induced thymic atrophy. A slight increase in the concentration of corticosterone was found in mice infected with the highly virulent strain, and thymocytes presented an increased susceptibility to dexamethasone-induced death during disseminated infection. The administration of an antagonist of glucocorticoid receptors partially reverted the infection-induced thymic atrophy. We observed a reduction in all thymocyte populations analyzed, including the earliest thymic precursors, suggesting a defect during thymic colonization by T cell precursors and/or during the differentiation of these cells in the bone marrow in addition to local demise of thymic cells. Our data suggest a complex picture underlying thymic atrophy during infection by M. avium with the participation of locally produced NO, endogenous corticosteroid activity, and reduced bone marrow seeding.

Leishmania mitochondrial peroxiredoxin plays a crucial peroxidase-unrelated role during infection: insight into its novel chaperone activity.

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx(-)) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx(-) was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx(-) were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47(phox-/-) and B6.RAG2(-/-) IFN-γ(-/-) mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx(-). A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx(-) were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.

CXCR3 Provides a Competitive Advantage for Retention of Mycobacterium tuberculosis-Specific Tissue-Resident Memory T Cells Following a Mucosal Tuberculosis Vaccine.

Mycobacterium tuberculosis is a major human pathogen, and new vaccines are needed to prevent transmission. Mucosal vaccination may confer protection against M. tuberculosis by stimulating tissue-resident memory (TRM) CD4+ T cells in the lungs. The chemokine receptor CXCR3 promotes lung recruitment of T cells, but its role in TRM development is unknown. This study demonstrates the recombinant influenza A virus vaccine PR8.p25, expressing the immunodominant M. tuberculosis T cell epitope p25, induces CXCR3 expression on p25-specific CD4+ T cells in the lungs so that the majority of vaccine-induced CD4+ TRM expresses CXCR3 at 6 weeks. However, CXCR3-/- mice developed equivalent antigen-specific CD4+ T cell responses to wild-type (WT) mice following PR8.p25, and surprisingly retained more p25-specific CD4+ TRM in the lungs than WT mice at 6 weeks. The adoptive transfer of CXCR3-/- and WT P25 T cells into WT mice revealed that the initial recruitment of vaccine-induced CD4+ T cells into the lungs was independent of CXCR3, but by 6 weeks, CXCR3-deficient P25 T cells, and especially CXCR3-/- TRM, were significantly reduced compared to CXCR3-sufficient P25 T cells. Therefore, although CXCR3 was not essential for CD4+ TRM recruitment or retention, it provided a competitive advantage for the induction of M. tuberculosis-specific CD4+ TRM in the lungs following pulmonary immunization.

Toll-like receptor 9 is required for full host resistance to Mycobacterium avium infection but plays no role in induction of Th1 responses.

To investigate the role of Toll-like receptor 9 (TLR9) in innate immunity to Mycobacterium avium, TLR9, TLR2, and MyD88 knockout (KO) mice were infected with this bacterium. Bacterial burdens were higher in the spleens, livers, and lungs of infected TLR9 KO mice than in those of C57BL/6 mice, indicating that TLR9 is required for efficient control of M. avium infection. However, TLR9 KO or TLR2 KO spleen cells displayed normal M. avium-induced tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) responses. This finding was confirmed by determining the number of splenic CD4(+) T cells producing IFN-γ by flow cytometry. Furthermore, TLR2 and MyD88, but not TLR9, played a major role in interleukin-12 and TNF-α production by M. avium-infected macrophages and dendritic cells (DCs). We also found that major histocompatibility complex class II molecule expression on DCs is regulated by TLR2 and MyD88 signaling but not by TLR9. Finally, lack of TLR9, TLR2, or MyD88 reduced the numbers of macrophages, epithelioid cells, and lymphocytes in M. avium-induced granulomas but only MyD88 deficiency affected the number of liver granulomas. In summary, our data demonstrated that the involvement of TLR9 in the control of M. avium infection is not related to the induction of Th1 responses.

Influenza A Virus Hemagglutinin Is Produced in Different Disulfide-Bonded States.

Aims: Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in influenza A virus infection, and we hypothesized that this role involved manipulation of disulfide bonds in HA. Results: Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high-energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile. We measured the redox state of influenza A virus HA disulfide bonds and their susceptibility to cleavage by vascular thiol isomerases. Using differential cysteine alkylation and mass spectrometry, we show that all six HA disulfide bonds exist in unformed states in ∼1 in 10 recombinant and viral surface HA molecules. Four of the six H1 and H3 HA bonds are cleaved by the vascular thiol isomerases, thioredoxin and protein disulphide isomerase, in recombinant proteins, which correlated with surface exposure of the disulfides in crystal structures. In contrast, viral surface HA disulfide bonds are impervious to five different vascular thiol isomerases. Innovation: It has been assumed that the disulfide bonds in mature HA protein are intact and inert. We show that all six HA disulfide bonds can exist in unformed states. Conclusion: These findings indicate that influenza A virus HA disulfides are naturally labile but not substrates for thiol isomerases when expressed on the viral surface.

Boosting BCG with recombinant influenza A virus tuberculosis vaccines increases pulmonary T cell responses but not protection against Mycobacterium tuberculosis infection.

The current Mycobacterium bovis BCG vaccine provides inconsistent protection against pulmonary infection with Mycobacterium tuberculosis. Immunity induced by subcutaneous immunization with BCG wanes and does not promote early recruitment of T cell to the lungs after M. tuberculosis infection. Delivery of Tuberculosis (TB) vaccines to the lungs may increase and prolong immunity at the primary site of M. tuberculosis infection. Pulmonary immunization with recombinant influenza A viruses (rIAVs) expressing an immune-dominant M. tuberculosis CD4+ T cell epitope (PR8-p25 and X31-p25) stimulates protective immunity against lung TB infection. Here, we investigated the potential use of rIAVs to improve the efficacy of BCG using simultaneous immunization (SIM) and prime-boost strategies. SIM with parenteral BCG and intranasal PR8-p25 resulted in equivalent protection to BCG alone against early, acute and chronic M. tuberculosis infection. Boosting BCG with rIAVs increased the frequency of IFN-γ-secreting specific T cells (p<0.001) and polyfunctional CD4+ T cells (p<0.05) in the lungs compared to the BCG alone, however, this did not result in a significant increase in protection against M. tuberculosis compared to BCG alone. Therefore, sequential pulmonary immunization with these rIAVs after BCG increased M. tuberculosis-specific memory T cell responses in the lung, but not protection against M. tuberculosis infection.

CXCR6-Deficiency Improves the Control of Pulmonary Mycobacterium tuberculosis and Influenza Infection Independent of T-Lymphocyte Recruitment to the Lungs.

T-lymphocytes are critical for protection against respiratory infections, such as Mycobacterium tuberculosis and influenza virus, with chemokine receptors playing an important role in directing these cells to the lungs. CXCR6 is expressed by activated T-lymphocytes and its ligand, CXCL16, is constitutively expressed by the bronchial epithelia, suggesting a role in T-lymphocyte recruitment and retention. However, it is unknown whether CXCR6 is required in responses to pulmonary infection, particularly on CD4+ T-lymphocytes. Analysis of CXCR6-reporter mice revealed that in naïve mice, lung leukocyte expression of CXCR6 was largely restricted to a small population of T-lymphocytes, but this population was highly upregulated after either infection. Nevertheless, pulmonary infection of CXCR6-deficient mice with M. tuberculosis or recombinant influenza A virus expressing P25 peptide (rIAV-P25), an I-Ab-restricted epitope from the immunodominant mycobacterial antigen, Ag85B, demonstrated that the receptor was redundant for recruitment of T-lymphocytes to the lungs. Interestingly, CXCR6-deficiency resulted in reduced bacterial burden in the lungs 6 weeks after M. tuberculosis infection, and reduced weight loss after rIAV-P25 infection compared to wild type controls. This was paradoxically associated with a decrease in Th1-cytokine responses in the lung parenchyma. Adoptive transfer of P25-specific CXCR6-deficient T-lymphocytes into WT mice revealed that this functional change in Th1-cytokine production was not due to a T-lymphocyte intrinsic mechanism. Moreover, there was no reduction in the number or function of CD4+ and CD8+ tissue resident memory cells in the lungs of CXCR6-deficient mice. Although CXCR6 was not required for T-lymphocyte recruitment or retention in the lungs, CXCR6 influenced the kinetics of the inflammatory response so that deficiency led to increased host control of M. tuberculosis and influenza virus.

PLGA particulate subunit tuberculosis vaccines promote humoral and Th17 responses but do not enhance control of Mycobacterium tuberculosis infection.

Tuberculosis places a staggering burden on human health globally. The new World Health Organisation End-TB Strategy has highlighted the urgent need for more effective TB vaccines to improve control of the disease. Protein-based subunit vaccines offer potential as safe and effective generators of protective immunity, and the use of particulate vaccine formulation and delivery by the pulmonary route may enhance local immunogenicity. In this study, novel particulate subunit vaccines were developed utilising biodegradable poly(lactic-co-glycolic acid) (PLGA) slow-release particles as carriers for the Mycobacterium tuberculosis lipoprotein MPT83, together with the adjuvants trehalose-dibehenate (TDB) or Monophosphoryl lipid A (MPL). Following delivery by the pulmonary or subcutaneous routes, the immunogenicity and protective efficacy of these vaccines were assessed in a murine model of M. tuberculosis infection. When delivered peripherally, these vaccines induced modest, antigen-specific Th1 and Th17 responses, but strong anti-MPT83 antibody responses. Mucosal delivery of the PLGA(MPT83) vaccine, with or without TDB, increased antigen-specific Th17 responses in the lungs, however, PLGA-encapsulated vaccines did not provide protection against M. tuberculosis challenge. By contrast, peripheral delivery of DDA liposomes containing MPT83 and TDB or MPL, stimulated both Th1 and Th17 responses and generated protection against M. tuberculosis challenge. Therefore, PLGA-formulated vaccines primarily stimulate strong humoral immunity, or Th17 responses if used mucosally, and may be a suitable carrier for vaccines against extracellular pathogens. This study emphasises the critical nature of the vaccine carrier, adjuvant and route of delivery for optimising vaccine efficacy against TB.

Pulmonary immunization with a recombinant influenza A virus vaccine induces lung-resident CD4+ memory T cells that are associated with protection against tuberculosis.

The lung is the primary site of infection with the major human pathogen, Mycobacterium tuberculosis. Effective vaccines against M. tuberculosis must stimulate memory T cells to provide early protection in the lung. Recently, tissue-resident memory T cells (TRM) were found to be phenotypically and transcriptional distinct from circulating memory T cells. Here, we identified M. tuberculosis-specific CD4+ T cells induced by recombinant influenza A viruses (rIAV) vaccines expressing M. tuberculosis peptides that persisted in the lung parenchyma with the phenotypic and transcriptional characteristics of TRMs. To determine if these rIAV-induced CD4+ TRM were protective independent of circulating memory T cells, mice previously immunized with the rIAV vaccine were treated with the sphingosine-1-phosphate receptor modulator, FTY720, prior to and during the first 17 days of M. tuberculosis challenge. This markedly reduced circulating T cells, but had no effect on the frequency of M. tuberculosis-specific CD4+ TRMs in the lung parenchyma or their cytokine response to infection. Importantly, mice immunized with the rIAV vaccine were protected against M. tuberculosis infection even when circulating T cells were profoundly depleted by the treatment. Therefore, pulmonary immunization with the rIAV vaccine stimulates lung-resident CD4+ memory T cells that are associated with early protection against tuberculosis infection.

Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium.

A panel of monoclonal antibodies specific for CD27 ligand (CD70), CD30 ligand (CD153), CD134 ligand (OX40L), and CD137 ligand (4-1BBL) were screened in vivo for their ability to affect the control of Mycobacterium avium infection in C57Bl/6 mice. Only the blocking of CD153 led to increased mycobacterial burdens. We then used CD30-deficient mice and found an increase in the proliferation of two strains of M. avium in these mice as compared with control animals. The increased mycobacterial growth was associated with decreased T cell expansion and reduced interferon-gamma (IFN-gamma) responses as a result of reduced polarization of the antigen-specific, IFN-gamma-producing T cells. At late times but not early in infection, the lymphoid cuff surrounding granulomas was depleted in the CD30-deficient animals. This report expands our knowledge about tumor necrosis factor superfamily members involved in the immune responses to mycobacterial infection by identifying CD30-CD153 interactions as required for optimal immune control of M. avium infection.

Delayed recruitment of lymphocytes into the lungs of CD30-deficient mice during aerogenic Mycobacterium avium infections.

CD30 is a member of the tumor necrosis factor-receptor superfamily, a group of receptors known to act as accessory molecules in the development of the immune response. Control and CD30-deficient mice were aerogenically infected with Mycobacterium avium. Although the mycobacterial loads in the lungs were similar in both strains of mice, CD30-deficient animals exhibited delayed structuring of pulmonary granulomas and reduced recruitment of lymphocytes throughout a 240 days period of infection. Discrete alterations in the chemokine network were detected in the CD30-deficient animals although they showed no clear relation to the deficient inflammatory response. Thus CD30/CD153 interactions are involved in lung immune-mediated inflammation.

Characterization of the deregulated immune activation occurring at late stages of mycobacterial infection in TNF-deficient mice.

In the absence of TNF, mice infected with Mycobacterium avium suffer a peculiar disintegration of the granulomas, with extensive apoptosis and necrosis of their cells, occurring during the course of the infection and leading to the death of the animals within a few days of its onset. The survival time depends on the virulence of the infecting strain as well as on the dose and route of infection. Intravenously infected mice developed the typical lesions in hepatic granulomas whereas aerosol-infected animals developed them in the lung granulomas. At the onset of the development of pulmonary granuloma disintegration, extensive expansion of T cells, with intense up-regulation of activation markers, massive exacerbation of their ability to secrete IFN-gamma, and increased cytotoxic activity of both CD4(+) and CD8(+) T cells were observed. Forced expression of Bcl2 did not prevent the early death of infected TNF-deficient mice leading merely to a modest increase in survival times. The expression of the FasL on T cells was not affected but there was an intense up-regulation of the TRAIL in T cells and, in particular, myeloid cells. We thus show that an exacerbated immune response occurs in TNF-deficient hosts during M. avium infections that leads to enhanced IFN-gamma production and late up-regulation of TRAIL which may contribute to granuloma disintegration.

Virulence of Mycobacterium avium in mice does not correlate with resistance to nitric oxide.

The growth in C57Bl/6 mice of seven distinct Mycobacterium avium strains was not exacerbated by the disruption of the inducible (type 2) nitric oxide synthase regardless of the virulence of the strain. The susceptibility of this panel of M. avium strains to reactive nitrogen intermediates in a cell-free system, namely the exposure to acidified nitrite or to the NO donor NOC-18, showed that there is no correlation between strain virulence and the corresponding minimal bactericidal and minimal inhibitory concentrations for those compounds determined in vitro.

Genetic control of immune-mediated necrosis of Mycobacterium avium granulomas.

Intravenous infection of C57BL/6 and C57BL/10 mice with low doses of a highly virulent strain of Mycobacterium avium (ATCC 25291) led to the development of granulomas that underwent necrosis. In contrast, neither BALB/c nor DBA/1 mice developed granuloma necrosis after such infection despite a similar course of mycobacterial proliferation. Studies with C57BL/10 mice congenic for the Hc locus revealed that an intact complement C5 gene is required for granuloma necrosis. On the other hand, genetic disruption of the interleukin-10 gene in BALB/c mice made this strain susceptible to granuloma necrosis.

Regulation of granuloma fibrosis by nitric oxide during Mycobacterium avium experimental infection.

Collagen deposition within granulomas formed after Mycobacterium avium infection was analysed on histological sections stained with Masson's trichrome using acquired computerized image analysis and a program that was specifically designed for that purpose. Comparison was made between immunocompetent C57BL/6 mice and mice genetically deficient in the inducible nitric oxide (NO) synthase gene (iNOS(-/-) mice) infected with either a highly virulent strain or a moderately virulent strain of M. avium. iNOS-deficient mice were more resistant to the highly virulent strain than control C57B1/6 mice, but both strains were equally susceptible to the less virulent M. avium strain. Collagen distribution in the granuloma was found in the cuff surrounding the granuloma in an area rich in lymphoid cells as well as inside the granuloma itself, conferring a mesh-like structure within that lesion. It was seen that iNOS(-/-) mice induced a higher collagen deposition than C57BL/6 mice and that such collagen deposition varied with the mycobacterial strain used to infect the animals.