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RationaleThe molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood.ObjectivesTo identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis.MethodsWe used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection.Measurements and main resultsWe identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2+ CD4+ T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection.ConclusionsTOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

CD1 proteins evolved to present diverse lipid Ags to T cells. In comparison with MHC proteins, CD1 proteins exhibit minimal allelic diversity as a result of nonsynonymous single nucleotide polymorphisms (SNPs). However, it is unknown if common SNPs in gene regulatory regions affect CD1 expression and function. We report surprising diversity in patterns of inducible CD1a expression on human dendritic cells (DCs), spanning the full range from undetectable to high density, a finding not seen with other CD1 isoforms. CD1a-deficient DCs failed to present mycobacterial lipopeptide to T cells but had no defects in endocytosis, cytokine secretion, or expression of costimulatory molecules after LPS treatment. We identified an SNP in the 5′ untranslated region (rs366316) that was common and strongly associated with low CD1a surface expression and mRNA levels (p = 0.03 and p = 0.001, respectively). Using a CD1a promoter-luciferase system in combination with mutagenesis studies, we found that the polymorphic allele reduced luciferase expression by 44% compared with the wild-type variant (p < 0.001). Genetic regulation of lipid Ag presentation by varying expression on human DCs provides a mechanism for achieving population level differences in immune responses despite limited structural variation in CD1a proteins.

RationaleUnderstanding mechanisms of resistance to M. tuberculosis (M.tb) infection in humans could identify novel therapeutic strategies as it has for other infectious diseases, such as HIV.ObjectivesTo compare the early transcriptional response of M.tb-infected monocytes between Ugandan household contacts of tuberculosis patients who demonstrate clinical resistance to M.tb infection (cases) and matched controls with latent tuberculosis infection.MethodsCases (n = 10) and controls (n = 18) were selected from a long-term household contact study in which cases did not convert their tuberculin skin test (TST) or develop tuberculosis over two years of follow up. We obtained genome-wide transcriptional profiles of M.tb-infected peripheral blood monocytes and used Gene Set Enrichment Analysis and interaction networks to identify cellular processes associated with resistance to clinical M.tb infection.Measurements and main resultsWe discovered gene sets associated with histone deacetylases that were differentially expressed when comparing resistant and susceptible subjects. We used small molecule inhibitors to demonstrate that histone deacetylase function is important for the pro-inflammatory response to in-vitro M.tb infection in human monocytes.ConclusionsMonocytes from individuals who appear to resist clinical M.tb infection differentially activate pathways controlled by histone deacetylase in response to in-vitro M.tb infection when compared to those who are susceptible and develop latent tuberculosis. These data identify a potential cellular mechanism underlying the clinical phenomenon of resistance to M.tb infection despite known exposure to an infectious contact.

Humans exposed to Mycobacterium tuberculosis (Mtb) have variable susceptibility to tuberculosis (TB) and its outcomes. Siglec-5 and Siglec-14 are members of the sialic-acid binding lectin family that regulate immune responses to pathogens through inhibitory (Siglec-5) and activating (Siglec-14) domains. The SIGLEC14 coding sequence is deleted in a high proportion of individuals, placing a SIGLEC5-like gene under the expression of the SIGLEC14 promoter (the SIGLEC14 null allele) and causing expression of a Siglec-5 like protein in monocytes and macrophages. We hypothesized that the SIGLEC14 null allele was associated with Mtb replication in monocytes, T-cell responses to the BCG vaccine, and clinical susceptibility to TB. The SIGLEC14 null allele was associated with protection from TB meningitis in Vietnamese adults but not with pediatric TB in South Africa. The null allele was associated with increased IL-2 and IL-17 production following ex-vivo BCG stimulation of blood from 10 week-old South African infants vaccinated with BCG at birth. Mtb replication was increased in THP-1 cells overexpressing either Siglec-5 or Siglec-14 relative to controls. To our knowledge, this is the first study to demonstrate an association between SIGLEC expression and clinical TB, Mtb replication, or BCG-specific T-cell cytokines.

Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individual's susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.

Pediatric tuberculosis (TB) is a significant cause of death in children and has a different pathogenesis than adult TB, but BCG-induced immunity is incomplete. Although genetic factors regulate BCG-induced immunity and TB susceptibility, these genes are mostly unknown and have largely been unexamined in pediatric TB. We hypothesized that TOLLIP, a TLR suppressor, orchestrates immune responses to M. tuberculosis (MTb) and susceptibility to pediatric TB. We discovered that a polymorphism of TOLLIP, rs5743854, regulated signaling in a promoter assay and was associated with TOLLIP mRNA deficiency in infant peripheral blood monocytes (p = 0.008, Kruskal-Wallis). The rs5743854 G allele was associated with protection from pediatric TB in a South African cohort (N=226 cases and 626 controls, dominant model, p = 0.002, odds ratio (OR) 0.63 unadjusted analysis; p = 0.011, OR = 0.58 adjusted for ethnicity by ancestry informative markers). The G allele was also associated with decreased frequency of BCG-specific CD4+IL-2 responses (p = 0.003, Kruskal-Wallis test) and CD4+ T-cell proliferation (p = 0.03, generalized linear model) in 174 infants vaccinated with BCG at birth and re-stimulated at 10 weeks of age. To examine mechanisms of protection, we created a TOLLIP-deficient THP-1 cell line. TOLLIP-deficient monocytes had increased MTb-induced TNF and reactive nitrogen secretion and reduced MTb replication compared to control cells. Together, these data suggest that TOLLIP deficiency is associated with enhanced anti-microbial monocyte responses that control MTb replication and provide protection against pediatric TB disease. Suppressing TOLLIP levels by host-directed therapeutics may provide adjunctive therapy for pediatric TB.

Most of household contacts of pulmonary TB cases, develop latent Mtb infection (LTBI). In a large TB household contact study in Uganda, we found that 9.1% of contacts remained persistently TST negative in 2 years of follow up. We hypothesized that resistance to Mtb infection is mediated by monocytes. We compared genome-wide mRNA profiles of Mtb-infected monocytes from those who are resistant or susceptible to Mtb infection. Using Gene Set Enrichment Analysis, we recently identified a butyrate stimulated gene set that distinguished these two groups. Butyrate inhibits histone deacetylases (HDAC), a gene family that regulates transcription as well as innate immune responses to microbes. We found that treatment of peripheral blood monocytes with class I HDAC inhibitors, depsipeptide and sodium butyrate, led to reduced IL6 and TNF secretion after Mtb infection compared to untreated cells. In contrast, IL1beta levels were increased. Together, these data indicate that class I HDAC inhibitors modulate proinflammatory cytokines after Mtb infection. Future studies will examine how HDAC inhibitors regulate inflammasome-dependent IL-1beta and IL6 secretion differently. HDACs likely regulate multiple pathways that modulate Mtb pathogenesis and mediate clinical to Mtb infection. HDACs are plausible candidate human Mtb resistance genes and promising targets for host-directed therapeutics.

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB., Author Summary Tuberculosis (TB) is one of the leading infectious causes of death worldwide. The current vaccine for TB, BCG, is widely used but it is not highly effective in preventing disease. We investigated the role of host genetics in the immune response to BCG vaccination. We found that variants of innate immunity genes (TLR1 and TLR6) were associated with BCG-induced immune responses after vaccination. These findings may guide new strategies for vaccine development as well as diagnosis of TB.

The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-β promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1(-/-) mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1(-/-) mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2(-/-) animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1(-/-) animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2(-/-) animals. Furthermore, the lungs of both Nod1(-/-) and Nod2(-/-) mice had significantly increased pro-inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.

Human cytomegalovirus (HCMV) infection of a cell containing latent Kaposi's sarcoma-associated herpesvirus (KSHV) results in the activation of KSHV lytic replication and the production of infectious virus. In this study, we examined the HCMV genes identified as having a role in the activation of HCMV early genes for their ability to activate KSHV lytic replication. It was found that the UL112-113 locus was able to activate the complete KSHV lytic cycle, while the UL122-123 locus, encoding the IE1 and IE2 proteins, known to be strong transactivators, did not.

Toll-like receptors (TLRs) are important regulators of the innate immune response to pathogens, including Mycobacterium leprae, which is recognized by TLR1/2 heterodimers. We previously identified a transmembrane domain polymorphism, TLR1_T1805G, that encodes an isoleucine to serine substitution and is associated with impaired signaling. We hypothesized that this TLR1 SNP regulates the innate immune response and susceptibility to leprosy. In HEK293 cells transfected with the 1805T or 1805G variant and stimulated with extracts of M. leprae, NF-κB activity was impaired in cells with the 1805G polymorphism. We next stimulated PBMCs from individuals with different genotypes for this SNP and found that 1805GG individuals had significantly reduced cytokine responses to both whole irradiated M. leprae and cell wall extracts. To investigate whether TLR1 variation is associated with clinical presentations of leprosy or leprosy immune reactions, we examined 933 Nepalese leprosy patients, including 238 with reversal reaction (RR), an immune reaction characterized by a Th1 T cell cytokine response. We found that the 1805G allele was associated with protection from RR with an odds ratio (OR) of 0.51 (95% CI 0.29–0.87, p = 0.01). Individuals with 1805 genotypes GG or TG also had a reduced risk of RR in comparison to genotype TT with an OR of 0.55 (95% CI 0.31–0.97, p = 0.04). To our knowledge, this is the first association of TLR1 with a Th1-mediated immune response. Our findings suggest that TLR1 deficiency influences adaptive immunity during leprosy infection to affect clinical manifestations such as nerve damage and disability., Author Summary Mycobacterium leprae (ML) causes a disabling and stigmatizing disease that is characterized by distinct immune responses. ML produces a spectrum of illness in humans, and several lines of evidence indicate that host genetic factors influence susceptibility and clinical manifestations. Leprosy can occur as the lepromatous or tuberculoid forms, which are associated with different clinical manifestations, histopathology, T cell cytokine profiles, and bacterial burden in affected sites. Leprosy is also associated with unique immunologic reactions, such as reversal reaction, which is characterized by the rapid development of a Th1 T cell cytokine response that can cause substantial morbidity. We and others recently discovered a common human polymorphism in TLR1 (T1805G, I602S) that regulates cytokine production in response to lipopeptide stimulation, influences the cellular innate immune response to Mycobacteria, is associated with altered localization, and is present in 50% of individuals worldwide. Here, we show that in humans the 1805G variant does not mediate an inflammatory response to ML in vitro and that this polymorphism is associated with protection from reversal reaction. These data suggest that a common variant of TLR1 is associated with altered adaptive immune responses to ML as well as clinical outcome.

Background Leprosy is characterized by polar clinical, histologic and immunological presentations. Previous immunologic studies of leprosy polarity were limited by the repertoire of cytokines known at the time. Methodology We used a candidate gene approach to measure mRNA levels in skin biopsies from leprosy lesions. mRNA from 24 chemokines and cytokines, and 6 immune cell type markers were measured from 85 Nepalese leprosy subjects. Selected findings were confirmed with immunohistochemistry. Principal Results Expression of three soluble mediators (CCL18, CCL17 and IL-10) and one macrophage cell type marker (CD14) was significantly elevated in lepromatous (CCL18, IL-10 and CD14) or tuberculoid (CCL17) lesions. Higher CCL18 protein expression by immunohistochemistry and a trend in increased serum CCL18 in lepromatous lesions was observed. No cytokines were associated with erythema nodosum leprosum or Type I reversal reaction following multiple comparison correction. Hierarchical clustering suggested that CCL18 was correlated with cell markers CD209 and CD14, while neither CCL17 nor CCL18 were highly correlated with classical TH1 and TH2 cytokines. Conclusions Our findings suggest that CCL17 and CCL18 dermal expression is associated with leprosy polarity., Author Summary Leprosy presents with a polarized spectrum, with lepromatous leprosy having high bacillary numbers and TH2 dermal cytokines, versus tuberculoid leprosy showing very few bacilli and TH1 cytokines. The mechanism underlying this polarized presentation is largely unknown. In the following study, we isolated mRNA from skin biopsies from 85 individuals with leprosy and measured the expression of a panel of 24 cytokines and 6 cell markers. We found that three soluble mediators (CCL17, CCL18 and IL10) and one cell marker (CD14) were differentially expressed in leprosy dermal lesions. CCL18 and IL10 were more highly expressed within lepromatous lesions, and CCL17 and CD14 were more highly expressed within tuberculoid lesions. In addition, CCL18 protein expression was confirmed by immunostaining. CCL17 and CCL18, were more strongly associated with leprosy polarity than traditional TH1 and TH2 cytokines. These data suggest that newer soluble chemokines may be important in leprosy pathogenesis and uncover a molecular signature of the two polar phenotypes of leprosy, which may be useful in future diagnostics.

An enzyme-based colorimetric assay for choline-containing phospholipids is described. When tested with egg yolk and serum, the method proved very sensitive, correlated well with other methods and avoided turbidity problems. The method is potentially applicable to a wide variety of samples.

An engineering evaluation of T-10 troop-type harnesses, risers, and chest reserve parachute assemblies of year classes (dates of manufacture) 1964 and 1965 was conducted by laboratory testing of component materials. The data obtained from limited samplings from Fort Bragg, No. Carolina, Alaska and the Panama Canal Zone indicated a generally serviceable condition for this equipment at nine and eight years of total age. Taken with more extensive data and with the technical background and criteria developed in the earlier basic report 72- 59-CE, (DDC AD 742-668) of the same title, dated March 1972, it was concluded that the recently extended 13 year (risers and harnesses) and 12 year (canopy assemblies) limits are fully substantiated for these 1964-65 classes. Subject to later check testing, further extension of the age life limit may be found appropriate.