Gregory W. Martens

Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome–dependent processing of IL-1β

Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

Hypercholesterolemia impairs immunity to tuberculosis.

ABSTRACT We demonstrate that apolipoprotein E -deficient (ApoE−/−) mice are highly susceptible to tuberculosis and that their susceptibility depends on the severity of hypercholesterolemia. Wild-type (WT) mice and ApoE−/− mice fed a low-cholesterol (LC) or high-cholesterol (HC) diet were infected with ∼50 CFU Mycobacterium tuberculosis Erdman by aerosol. ApoE−/− LC mice were modestly more susceptible to tuberculosis than WT LC mice. In contrast, ApoE−/− HC mice were extremely susceptible, as evidenced by 100% mortality after 4 weeks with tuberculosis. The lung pathology of ApoE−/− HC mice was remarkable for giant abscess-like lesions, massive infiltration by granulocytes, elevated inflammatory cytokine production, and a mean bacterial load ∼2 log units higher than that of WT HC mice. Compared to WT HC mice, the gamma interferon response of splenocytes restimulated ex vivo with M. tuberculosis culture filtrate protein was delayed in ApoE−/− HC mice, and they failed to control M. tuberculosis growth in the lung. OT-II cells adoptively transferred into uninfected ApoE−/− HC mice had a weak proliferative response to their antigen, indicating impaired priming of the adaptive immune response. Our studies show that ApoE−/− deficiency is associated with delayed expression of adaptive immunity to tuberculosis caused by defective priming of the adaptive immune response and that elevated serum cholesterol is responsible for this effect.

Diabetic Mice Display a Delayed Adaptive Immune Response to Mycobacterium tuberculosis

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. We previously reported that streptozotocin-induced DM impaired TB defense in mice, resulting in higher pulmonary bacterial burden, more extensive inflammation, and higher expression of several proinflammatory cytokines known to play a protective role in TB. In the current study, we tested the hypothesis that DM leads to delayed priming of adaptive immunity in the lung-draining lymph nodes (LNs) following low dose aerosol challenge with virulent Mycobacterium tuberculosis. We show that M. tuberculosis-specific IFN-γ–producing T cells arise later in the LNs of diabetic mice than controls, with a proportionate delay in recruitment of these cells to the lung and stimulation of IFN-γ–dependent responses. Dissemination of M. tuberculosis from lung to LNs was also delayed in diabetic mice, although they showed no defect in dendritic cell trafficking from lung to LNs after LPS stimulation. Lung leukocyte aggregates at the initial sites of M. tuberculosis infection developed later in diabetic than in nondiabetic mice, possibly related to reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early time points postinfection. We conclude that TB increased susceptibility in DM results from a delayed innate immune response to the presence of M. tuberculosis-infected alveolar macrophages. This in turn causes late delivery of Ag-bearing APC to the lung draining LNs and delayed priming of the adaptive immune response that is necessary to restrict M. tuberculosis replication.

CD4 T cell-mediated heterologous immunity between mycobacteria and poxviruses

ABSTRACT The bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis is used in many parts of the world as a vaccine against Mycobacterium tuberculosis. Some epidemiological evidence has suggested that BCG immunization may have unpredicted effects on resistance to other pathogens. We show here in a mouse model that BCG immunization followed by antibiotic treatment to clear the host of the pathogen rendered three strains of mice partially resistant to infection with vaccinia virus (VV) but not to lymphocytic choriomeningitis virus (LCMV). VV-challenged BCG-immune mice developed a striking splenomegaly and elevated CD4 and CD8 T-cell responses by 6 days postinfection (p.i.). However, resistance to VV infection could be seen as early as 1 to 2 days p.i. and was lost after antibody depletion of CD4 T-cell populations. BCG- but not LCMV-immune memory phenotype CD4 T cells preferentially produced gamma interferon (IFN-γ) in vivo after VV challenge. In contrast, LCMV-immune CD8 T cells preferentially produced IFN-γ in vivo in response to VV infection. In BCG-immune mice the resistance to VV infection and VV-induced CD4 T-cell IFN-γ production were ablated by cyclosporine A, which inhibits signaling through the T-cell receptor. This study therefore demonstrates CD4 T-cell-mediated heterologous immunity between a bacterium and virus. Further, it poses the question of whether BCG immunization of humans alters resistance to unrelated pathogens.

Pro-IL-16 Regulation in Activated Murine CD4+ Lymphocytes

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4+ T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G0/G1 arrest by stabilizing the cyclin-dependent kinase inhibitor p27Kip1. In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27Kip1 were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4+ T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4+ T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4+ T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.

Hypercholesterolemic LDL receptor-deficient mice mount a neutrophilic response to tuberculosis despite the timely expression of protective immunity.

The prevalence of hypercholesterolemia is rising in industrialized and developing countries. We reported previously that host defense against Mtb was impaired by hypercholesterolemia in ApoE−/− mice, raising the possibility that people with HC could be more vulnerable to TB. The present study examined whether TB immunity was similarly impaired in a different hypercholesterolemic model, LDL‐R−/− mice, which developed comparable elevation of total serum cholesterol as ApoE−/−mice when fed HC or LC diets. Like ApoE−/− mice, LDL‐R−/− mice had an exaggerated lung inflammatory response to Mtb with increased tissue necrosis. Inflammation, foamy macrophage formation, and tissue necrosis in LDL‐R−/− mice increased with the degree of hypercholesterolemia. Unlike ApoE−/− mice, LDL‐R−/− mice fed a HC diet mounted a timely and protective adaptive immune response that restricted mycobacterial replication comparably with WT mice. Thus, ApoE−/− and LDL‐R−/− mice share a cholesterol‐dependent hyperinflammatory TB phenotype but do not share the impairment of adaptive immunity found in ApoE−/− mice. The impact of hypercholesterolemia on TB immunity is more complex than appreciated by total cholesterol alone, possibly reflecting the different functional effect of specific lipoprotein particles.

Initiation of Acquired Immunity in the Lungs of Mice Lacking Lymph Nodes after Infection with Aerosolized Mycobacterium tuberculosis

Recent evidence points to lung draining lymph nodes as the site that initiates the immune response in mice infected with aerosolized Mycobacterium tuberculosis. Here we expanded these studies and showed that infection of mice that lack lymph nodes with aerosolized M. tuberculosis results in a massive mononuclear cell infiltrate in the lungs within 14 days postinfection. This infiltration clearly resembles an expansion of the bronchus-associated lymphoid tissue. As expected, no bronchus-associated lymphoid tissue was observed in M. tuberculosis-infected wild-type control mice. Importantly, acquired specific immune response to M. tuberculosis antigens could be detected in lung lymphocytes harvested from mice lacking lymph nodes as early as 14 days postinfection. In addition, the bacterial burden in these mice was indistinguishable from that observed in wild-type C57BL/6 control mice. These results indicate that in the absence of lymph nodes, priming of the immune response occurs in the lung tissues after infection of mice with aerosolized M. tuberculosis and clearly illustrate the enormous plasticity of the immune system to develop resistance to foreign pathogens.

Chromatin Decondensation and T Cell Hyperresponsiveness in Diabetes-Associated Hyperglycemia

Diabetes is linked to increased inflammation and susceptibility to certain infectious diseases including tuberculosis (TB). We previously reported that aerosol TB in mice with chronic (≥12 wk) hyperglycemia features increased bacterial load, overproduction of several cytokines, and increased immune pathology compared with normoglycemic controls. A similar phenotype exists in human patients with diabetes with TB. The mechanisms of increased T cell activation in diabetes are unknown. In the current study, we tested the hypothesis that hyperglycemia modifies the intrinsic responsiveness of naive T cells to TCR stimulation. Purified T cells from chronically hyperglycemic (HG) mice produced higher levels of Th1, Th2, and Th17 cytokines and proliferated more than T cells from normoglycemic controls after anti-CD3e or Ag stimulation. In this way, naive T cells from HG mice resembled Ag-experienced cells, although CD44 expression was not increased. Chromatin decondensation, another characteristic of Ag-experienced T cells, was increased in naive T cells from HG mice. That phenotype depended on expression of the receptor for advanced glycation end products and could be reversed by inhibiting p38 MAPK. Chromatin decondensation and hyperresponsiveness to TCR stimulation persisted following transfer of T cells from HG mice into normoglycemic mice. We propose that chronic hyperglycemia causes receptor for advanced glycation end products–mediated epigenetic modification of naive T cells leading to p38 MAPK-dependent chromatin decondensation. This preactivation state facilitates transcription factor access to DNA, increasing cytokine production and proliferation following TCR stimulation. This mechanism may contribute to pathological inflammation associated with diabetes and might offer a novel therapeutic target.

Is Hypercholesterolemia a Friend or a Foe of Tuberculosis

) mice are highly susceptibleto tuberculosis (TB) and that this susceptibility depends on theseverity of hypercholesterolemia. However, it may be morereasonable for the authors to stress that ApoE deficiency itself,rather than hypercholesterolemia, is a critical risk factor forTB. Low-density lipoprotein (LDL) receptor-deficient(LDLR

Defects in early cell recruitment contribute to the increased susceptibility to respiratory Klebsiella pneumoniae infection in diabetic mice.

Diabetes is associated with increased susceptibility to Klebsiella pneumoniae and poor prognosis with infection. We demonstrate accelerated mortality in mice with streptozotocin-induced diabetes following tracheal instillation of K. pneumoniae. Diabetic mice recruited fewer granulocytes to the alveolar airspace and had reduced early production of CXCL1, CXCL2, IL-1β and TNF-α following tracheal instillation of K. pneumoniae-lipopolysaccharide. Additionally, TLR2 and TIRAP expression following K. pneumoniae-lipopolysaccharide exposure was decreased in hyperglycemic mice. These findings indicate that impaired innate sensing and failure to rapidly recruit granulocytes to the site of infection is a mechanism for diabetic susceptibility to respiratory K. pneumoniae infection.