Immune privilege of the CNS is not the consequence of limited antigen sampling.

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.

Inflammatory dendritic cells migrate in and out of transplanted chronic mycobacterial granulomas in mice.

An estimated one-third of the world's population is infected with Mycobacterium tuberculosis, although most affected individuals maintain a latent infection. This control is attributed to the formation of granulomas, cell masses largely comprising infected macrophages with T cells aggregated around them. Inflammatory DCs, characterized as CD11c+CD11b+Ly6C+, are also found in granulomas and are an essential component of the acute immune response to mycobacteria. However, their function during chronic infection is less well understood. Here, we report that CD11c+ cells dynamically traffic in and out of both acute and chronic granulomas induced by Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) in mice. By transplanting Mycobacterium-induced granulomas containing fluorescently labeled CD11c+ cells and bacteria into unlabeled mice, we were able to follow CD11c+ cell trafficking and T cell activation. We found that half of the CD11c+ cells in chronic granulomas were exchanged within 1 week. Compared with tissue-resident DC populations, CD11c+ cells migrating out of granuloma-containing tissue had an unexpected systemic dissemination pattern. Despite low antigen availability, systemic CD4+ T cell priming still occurred during chronic infection. These data demonstrate that surveillance of granulomatous tissue by CD11c+ cells is continuous and that these cells are distinct from tissue-resident DC populations and support T cell priming during both stages of Mycobacterium infection. This intense DC surveillance may also be a feature of Mycobacterium tuberculosis infection and other granuloma-associated diseases.

Granuloma formation and host defense in chronic Mycobacterium tuberculosis infection requires PYCARD/ASC but not NLRP3 or caspase-1.

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1beta secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3(-/-), Casp-1(-/-), and WT mice showed no differences in pulmonary IL-1beta production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard(-/-) mice. Decreased survival of Pycard(-/-) animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.

Dendritic cells in chronic mycobacterial granulomas restrict local anti-bacterial T cell response in a murine model.

BACKGROUND: Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c(+) cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c(+) cells from acute granulomas. As a consequence of their phenotype, CD11c(+) cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4(+)IFNgamma(+) T cells or induce an IFNgamma response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c(+) cells from chronic lesions to stimulate a protective IFNgamma T cell response. CONCLUSIONS/SIGNIFICANCE: Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.

Intracerebral Mycobacterium bovis bacilli Calmette-Guerin infection-induced immune responses in the CNS.

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-gamma(+)CD4(+) T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-gammaIL-17 double-positive CD4(+) T cells is unique to the CNS. The major CNS source of TNF-alpha is microglia, with modest production by CD4(+) T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3(+)CD4(+) T cells and PD-L2(+) dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection.

Requirements for CD4(+) T cell levels in acute Mycobacterium bovis strain bacille Calmette Guerin (BCG)-induced granulomas differ for optimal mycobacterial control versus granuloma formation.

Bacille Calmette Guérin (BCG)-induced granulomas contain T cells that express a broad TCR repertoire even at the level of the individual lesion. We have developed a BCG infection model in mice having only one T cell specific for a recombinant BCG epitope expressed in a lipoprotein fusion protein. Here we report that the single T cell model induces well-formed granulomas, but has weaker protection than that conferred by wild-type granulomas. This finding correlates with lower CD4(+) T cell recruitment into acute granulomas (3 weeks post infection). Chronic granulomas (6 weeks post infection) contain similar proportions of CD4(+) T cells in both models, but in the single T cell model the proportion of leukocyte function-associated antigen-1 low, non-IFNgamma-producing CD4(+) T cells is lower. In fact, even though it is likely that there are very few, if any, IFNgamma(+) CD4(+) T cells present in the single T cell model, granuloma integrity is not influenced, indicating that high levels of IFNgamma are not required for granuloma maintenance. These data underline the importance of early CD4(+) T cell recruitment into the granuloma to anti-mycobacterial protection and show that CD4(+) T cell levels required for granuloma formation and optimal protection are different. These data also show that T cell repertoire complexity contributes to protection against mycobacteria.