CXCR5 EXPRESSING T HELPER CELLS MEDIATE PROTECTIVE IMMUNITY AGAINST TUBERCULOSIS

One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we show that organized ectopic lymphoid structures containing CXCR5+ T cells are found in Mtb-infected lungs. In addition, we show that in experimental Mtb infection models, the presence of CXCR5+ T cells inside ectopic lymphoid structures are associated with immune control. Furthermore, in a mouse model of Mtb infection, we show that activated CD4+ CXCR5+ T cells accumulate in Mtb-infected lungs, and produce proinflammatory cytokines. Absence of CXCR5 in mice results in increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We show that CXCR5 expression on T cells mediates correct T cell localization within TB granulomas, efficient macrophage activation, promoting protection against Mtb infection and facilitating lymphoid follicle formation. These data show a novel role for CD4+ CXCR5+ T cells in protective immunity against TB and highlight their potential use for future TB vaccine design and therapy

Unexpected role for IL-17 in protective immunity against hypervirulent Mycobacterium tuberculosis HN878 infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world's population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered "hypervirulent" as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains. © 2014 Gopal et al

S100A8/A9 Proteins Mediate Neutrophilic Inflammation and Lung Pathology during Tuberculosis

Rationale: A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined. Objectives: The objective of this study was to identify the underlying immune mechanisms involved in formation of inflammatory granulomas seen during active TB. Methods: The immune mediators involved in inflammatory granuloma formation during TB were assessed using human samples and experimental models of Mycobacterium tuberculosis infection, using molecular and immunologic techniques. Measurements and Main Results: We demonstrate that in human patients with active TB and in nonhuman primate models of M. tuberculosis infection, neutrophils producing S100 proteins are dominant within the inflammatory lung granulomas seen during active TB. Using the mouse model of TB, we demonstrate that the exacerbated lung inflammation seen as a result of neutrophilic accumulation is dependent on S100A8/A9 proteins. S100A8/A9 proteins promote neutrophil accumulation by inducing production of proinflammatory chemokines and cytokines, and influencing leukocyte trafficking. Importantly, serum levels of S100A8/A9 proteins along with neutrophil-associated chemokines, such as keratinocyte chemoattractant, can be used as potential surrogate biomarkers to assess lung inflammation and disease severity in human TB. Conclusions: Our results thus show a major pathologic role for S100A8/A9 proteins in mediating neutrophil accumulation and inflammation associated with TB. Thus, targeting specific molecules, such as S100A8/A9 proteins, has the potential to decrease lung tissue damage without impacting protective immunity against TB

Adenoviral IL-17 overexpression reverses the increased susceptibility to <i>Mtb</i> HN878 infection in IL-17^−/− mice.

<p>B6 or IL-17^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and were either infected with a vector control adenovirus expressing luciferase (Adluc) or with an adenoviral vector overexpressing IL-17 (AdIL-17) on day 9. Lung bacterial burden was determined on D30 post-<i>Mtb</i> infection (a). Pulmonary histology was assessed on formalin-fixed, paraffin embedded lung sections that were stained with H&E (b). 100× magnification for H&E sections. The average size of T cell perivascular cuffing (c) and B cell lymphoid follicles (d) was calculated using the morphometric tool of the Zeiss Axioplan microscope. CXCL13 mRNA expression in formalin fixed, paraffin embedded lung sections was studied by in situ hybridization (e). 100× magnification, arrows indicate areas of CXCL-13 mRNA expression. The area occupied by CXCL13 signal by in situ hybridization per 200× field was calculated using the morphometric tool of the Zeiss Axioplan microscope. Serial sections from infected lungs were also processed for immunofluorescence using antibodies specific for CXCL13 and B220 (f). 400× magnification. Immunofluorescence staining was also performed using antibodies specific for or iNOS and F4/80 and the numbers of iNOS⁺ cells were counted (g). 400× magnification. The data points represent values from n = 5 mice per group. *p≤0.05, **p≤0.005, ***p≤0.0005, ns-not significant.</p

S100A8/A9 Proteins Mediate Neutrophilic Inflammation and Lung Pathology during Tuberculosis

Rationale: A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined. Objectives: The objective of this study was to identify the underlying immune mechanisms involved in formation of inflammatory granulomas seen during active TB. Methods: The immune mediators involved in inflammatory granuloma formation during TB were assessed using human samples and experimental models of Mycobacterium tuberculosis infection, using molecular and immunologic techniques. Measurements and Main Results: We demonstrate that in human patients with active TB and in nonhuman primate models of M. tuberculosis infection, neutrophils producing S100 proteins are dominant within the inflammatory lung granulomas seen during active TB. Using the mouse model of TB, we demonstrate that the exacerbated lung inflammation seen as a result of neutrophilic accumulation is dependent on S100A8/A9 proteins. S100A8/A9 proteins promote neutrophil accumulation by inducing production of proinflammatory chemokines and cytokines, and influencing leukocyte trafficking. Importantly, serum levels of S100A8/A9 proteins along with neutrophil-associated chemokines, such as keratinocyte chemoattractant, can be used as potential surrogate biomarkers to assess lung inflammation and disease severity in human TB. Conclusions: Our results thus show a major pathologic role for S100A8/A9 proteins in mediating neutrophil accumulation and inflammation associated with TB. Thus, targeting specific molecules, such as S100A8/A9 proteins, has the potential to decrease lung tissue damage without impacting protective immunity against TB

<i>Mtb</i> HN878-driven IL-17 production is dependent on TLR-2 and IL-1β.

<p>1×10⁶ lung DCs from B6 mice were infected in vitro with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878 (MOI 0.01) for 48 h and IL-1β production was determined in the supernatants (a). 1×10⁶ B6 DCs were infected with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878 (MOI 0.01), following which naïve B6 or IL-1R−/− total lung cell suspensions were added to the culture, and IL-17 levels were determined by ELISA on day 6 of the co-culture (b). B6 or IL-1R^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and lung bacterial burden was determined on D30 post-infection (c). The number of ESAT-6_1–20-specific, IL-17-producing cells in the lungs of uninfected (Un), <i>Mtb</i> HN878-infected B6 and IL-1R^−/− mice was determined by ELISpot assay (d). 1×10⁶ BMDCs were treated in vitro with whole cell lysate, cell wall extract or a lipid extract (20 µg/ml) of <i>Mtb</i> H37Rv or <i>Mtb</i> HN878, and IL-1β production in cell culture supernatants was determined by ELISA (e). 1×10⁶ lung CD11c⁺ cells from B6 or TLR-2^−/− mice and infected in vitro with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878. Subsequently, naïve B6 or TLR-2^−/− total lung cell suspensions were added to the culture, and IL-1β (f) and IL-17 (g) levels were determined by ELISA. The data points represent the mean (±SD) of values from 3–5 samples. *p≤0.05, **p≤0.005, ***p≤0.0005. nd-not detected.</p