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

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.

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.

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-gamma-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-gamma-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.

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-gamma) in vivo after VV challenge. In contrast, LCMV-immune CD8 T cells preferentially produced IFN-gamma in vivo in response to VV infection. In BCG-immune mice the resistance to VV infection and VV-induced CD4 T-cell IFN-gamma 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.

Hypercholesterolemia impairs immunity to tuberculosis.

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 approximately 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 approximately 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.

Tuberculosis susceptibility of diabetic mice.

Increased susceptibility to infections, including tuberculosis (TB), is a major cause of morbidity and mortality in patients with diabetes. Despite the clinical importance of this problem, little is known about how diabetes impairs protective immunity. We modeled this phenomenon by infecting acute (< or = 1 mo) or chronic (> or = 3 mo) diabetic mice with a low aerosol dose of Mycobacterium tuberculosis (Mtb) Erdman. Diabetes was induced by streptozotocin (STZ) treatment of C57BL/6 mice, while another mouse strain and diabetes model were used to confirm key observations. Lungs from acute diabetic and euglycemic mice had similar bacterial burdens, cytokine expression profiles, and histopathology. In contrast, chronic diabetic mice had > 1 log higher bacterial burden and more inflammation in the lung compared with euglycemic mice. The expression of adaptive immunity was delayed in chronic diabetic mice, shown by reduced early production of IFN-gamma in the lung and by the presence of fewer Mtb antigen (ESAT-6)-responsive T cells compared with euglycemic mice within the first month of infection. However, after 2 months of TB disease proinflammatory cytokines levels were higher in chronic diabetic than euglycemic mice. Here we show that Mtb infection of STZ-treated mice provides a useful model to study the effects of hyperglycemia on immunity. Our data indicate that the initiation of adaptive immunity is impaired by chronic hyperglycemia, resulting in a higher steady-state burden of Mtb in the lung.

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-1ß 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.

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.

Swine leukocyte antigen (SLA) diversity in Sinclair and Hanford swine.

The swine leukocyte antigen (SLA) haplotype B is associated with increased penetrance of the tumor traits in Sinclair swine cutaneous melanoma (SSCM). We established a series of SinclairxHanford swine crosses to facilitate genetic mapping of the tumor-associated loci. In this study, the SLA diversity in the founding animals was characterized for effective selection of maximum tumor penetrance in the pedigrees. Using the sequence-based typing (SBT) method we identified a total of 29 alleles at five polymorphic SLA loci (SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing six class I and five class II haplotypes. We subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the crossbred progeny. In a total of 469 animals we identified three crossovers within the class I region and three between the class I and class II regions, which corresponded to recombination frequencies of 0.39% and 0.56%, respectively. We also confirmed the presence of two expressed SLA-1 loci in three of the class I haplotypes and were able to determine the relative chromosomal arrangement of the duplicated loci in two haplotypes. This study furthers our understanding of the allelic architecture and polymorphism of the SLA system and will facilitate the mapping of loci associated with the expression of SSCM.

Characterization of swine leukocyte antigen polymorphism by sequence-based and PCR-SSP methods in Meishan pigs.

Resource herds of swine leukocyte antigen (SLA)-characterized pigs are an important tool for the study of immune responses, disease resistance, and production traits. They are also valuable large animal models for biomedical research, such as transplantation. The Meishan breed of pig is an economically significant breed that is available at several research institutions in the United States. We have characterized the SLA polymorphism of the breeding stock in the herd maintained at the University of Illinois and developed a simple assay to SLA type individuals within that herd. We have used a reverse transcription-polymerase chain reaction (RT-PCR)-based SLA typing method to clone and DNA sequence 19 SLA alleles at three SLA class Ia (SLA-1, SLA-2, and SLA-3) and two SLA class II (SLA-DRB1 and SLA-DQB1) loci. Based on this sequence information, a rapid SLA typing assay was developed to discriminate each allele using PCR with sequence-specific primers (PCR-SSP). Using this method, we were able to characterize the entire Meishan breeding stock and identify four SLA haplotypes present in the herd. The combination of SLA typing by cloning and DNA sequencing with PCR-SSP is therefore a valuable tool for the characterization of SLA alleles and haplotypes in resource herds of pigs.

DNA sequence based typing of swine leukocyte antigens in Yucatan miniature pigs.

BACKGROUND: We have established a breeding program to develop additional lines of swine leukocyte antigens (SLA) homozygous miniature pigs derived from the Yucatan Miniature Pig. Yucatan pigs have been used extensively in biomedical research since the 1970s and are known for their docile nature and small size. The breed has no consanguinity with the Hormel or Pittman-Moore breeds used to produce the NIH Miniature Pigs, the only other SLA homozygous miniature pigs in the USA. METHODS: SLA typing was initially done by restriction fragment length polymorphism. Then a cDNA library was constructed using spleen cells from these pigs, from which we cloned and sequenced nearly all alleles for the SLA-1, 3, 2, 6, DRB1, DQA and DQB1 loci. RESULTS: Four SLA homozygous lines were established with haplotypes that we have designated 'w, x, y and z'. An SLA class I/II crossover haplotype, designated 'q', with the SLA class I alleles of 'w' and the SLA class II alleles of 'z' was discovered and used to establish a fifth line. The cDNA sequences were used to develop locus specific primers for each locus and an reverse transcription-polymerase chain reaction sequence based typing (SBT) method. We have used this method to perform SBT on SLA homozygous Yucatan pigs with these haplotypes and on the NIH pig 'a, c and d' haplotypes. CONCLUSIONS: These pig lines represent a new resource for transplantation research and the methods we describe can be used to SLA type any herd of pigs.

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay.

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.