Microenvironments in Tuberculous Granulomas Are Delineated by Distinct Populations of Macrophage Subsets and Expression of Nitric Oxide Synthase and Arginase Isoforms

Abstract

Macrophages in granulomas are both anti-mycobacterial effector and host cell for Mycobacterium tuberculosis(M.tb), yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial nitric oxide synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared to non-granulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, while epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68 and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1 and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS:Arg1 expression in epithelioid macrophages, as compared to cells in the lymphocyte cuff. iNOS, Arg1 and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas while the inner regions were more likely to contain macrophages with pro-inflammatory, presumably bactericidal, phenotypes. Together these data support the concept that granulomas have organized microenvironments that balance anti-microbial anti-inflammatory responses to limit pathology in the lungs.

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16+ monocyte population via the IL-10/STAT3 axis

“…The NHP tissue samples were derived from BCG (Bacillus CalmetteGuérin) vaccinated and unvaccinated Mtb-infected rhesus macaques, exhibiting different degrees of lung pathology that was in general inversely correlated with time to end point (survival; Supplementary information, Figure S5A). Immunohistochemical analyses revealed that macrophage infiltration (CD68 + ) increased according to lung pathology severity ( Figure 4A, Supplementary information, Figure S5B); this pattern was also accompanied by increased CD163 expression in tuberculous granulomas in NHPs ( Figure 4A, Supplementary information, Figure S5B) [42]. Concomitantly, we observed an elevated expression of CD16 and MerTK in NHP tuberculous granulomas ( Figure 4A, Supplementary information, Figure S5B).…”

“…Considering that the CD16 + population expansion displays an M2-like phenotype in the pleural cavity from TB patients, and that TB pleural effusion activates STAT3 phosphorylation in monocytes from healthy donors and stimulates their protease-dependent migration, we infer that the environmental signals within Mtb infection sites perpetuate the CD16 + CD163 + MerTK + pSTAT3 + activation program and possibly also serve as chemoattractant. At the tissue level, although there are key reports integrating the concept of macrophage activation within tuberculous granulomas [41,42], to our knowledge this is the first study to identify the CD16 + CD163 + MerTK + macrophage activation program along with the co-localization of activated STAT3. Beyond the detection of CD163, we now provide a novel marker signature composed of CD16, MerTK and activated STAT3, which could be helpful to identify the anti-inflammatory and immunomodulatory macrophage program in the progression of TB disease.…”

Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas

“…The predominance of either enzyme spatially influences macrophage activation in different granuloma environments (8,(10)(11)(12)(13). In the presence of oxygen, NOS2 metabolizes L-arginine into L-citrulline and nitric oxide (NO), which is associated with killing of intracellular pathogens (10,(13)(14)(15).…”

Nitrite produced byMycobacterium tuberculosisin human macrophages in physiologic oxygen impacts bacterial ATP consumption and gene expression

“…The high-output pathway of RNS production by host inducible nitric oxide synthase (iNOS) contributes to control of diverse infections (1,2), including tuberculosis (TB) (3,4), via impacts on both the pathogen and the host (5). Macrophages in the lungs of humans (6,7) and macaques (8) with TB express functional iNOS. Correlative evidence suggests that the action of iNOS may contribute to human control of TB (1,3,9,10).…”