Host gene-encoded severe lung TB: from genes to the potential pathways.

We are reporting that the two-locus genotype -2518 macrophage chemoattractant protein (MCP)-1 GG and -1607 matrix metalloproteinase (MMP)-1 2G/2G promotes the expression of hyperinflammation in response to Mycobacterium tuberculosis infection, inducing extensive tissue damage and severe tuberculosis (TB) disease. Carriers of this two-locus genotype have a 13-fold higher chance of developing severe disease and 6.5-fold higher chance of developing permanent lesions, and a 3.864-fold higher chance of delayed response to first-line standardized treatment than carriers of any other relevant combination of genotypes at those two loci. Thus, these persons have an increased likelihood of poor health-related quality of life and of transmitting M. tuberculosis to other members of the community. In addition, through the analysis of human lung tissues, serum/plasma and in vitro experiments, including in vitro infections of THP-1 cells with M. tuberculosis and microarray analysis, we determined that this hyperinflammation state is potentially driven by the MCP-1/MMP-1/PAR-1 pathway. Hence, we are providing markers for the identification of TB cases that may develop severe pulmonary disease and delayed response to treatment, and are providing the basis for development of novel host-targeted clinical interventions to ameliorate the severity of pulmonary TB.

CCL2−2518 (A/G) polymorphisms and tuberculosis susceptibility: a meta-analysis.

BACKGROUND: It has been found that the -2518 C-C motif ligand (CCL)-2 promoter variant increases the risk of developing active tuberculosis (TB). OBJECTIVE: To study the association between -2518 variants and susceptibility to TB. DESIGN: We searched Medline, PubMed and the Wan Fang databases for human genetic studies on whether the -2518 CCL2 polymorphism influences the expression of active TB. Articles published from January 1998 to November 2010 were included. A random effects model was conducted in the meta-analysis. RESULTS: The CCL2-2518G allele (OR 1.51, 95%CI 1.11-2.04, P = 0.008) showed significant association with susceptibility to TB. In genotype analysis, the recessive model (CCL2 genotype GG, OR 1.66, 95%CI 1.19-2.33, P = 0.003) was slightly superior to the dominant model (G carrier genotypes OR 1.53, 95%CI 1.07-2.17, P = 0.018). These observations were prominent among Asians and Latin-Americans of Hispanic ancestry, but not in Africans from Ghana and South Africa. The presence of epistatic genes in one population but not in the other, environmental differences and pathogen virulence may account for this. CONCLUSION: The CCL2-2518G allele increases the risk of developing TB in Asians and Hispanics.

HLA-DRB1 alleles encoding the "shared epitope" are associated with susceptibility to developing rheumatoid arthritis whereas HLA-DRB1 alleles encoding an aspartic acid at position 70 of the beta-chain are protective in Mexican Mestizos.

The risk to develop rheumatoid arthritis (RA) has been associated with the presence of HLA-DRB1 alleles encoding the "shared epitope" (SE). Additionally, HLA-DRB1 alleles encoding an aspartic acid at position 70 (D70+ ) have been associated with protection against the development of RA. In this study we tested the association between either SE or D70+ and rheumatoid arthritis in Mexican Mestizos. We included 84 unrelated Mexican Mestizos patients with RA and 99 unrelated healthy controls. The HLA-typing was performed by PCR-SSO and PCR-SSP. We used the chi-squared test to detect differences in proportions of individuals carrying at least one SE or D70+ between patients and controls. We found that the proportion of individuals carrying at least one HLA-DRB1 allele encoding the SE was significantly increased in RA cases as compared to controls (p(c) = 0.0004, OR = 4.1, 95% CI = 2.2-7.7). The most frequently occurring allele was HLA-DRB1*0404 (0.161 vs 0.045). Moreover, we observed a significantly increased proportion of HLA-DRB1 SE+ cases with RF titers above the median (p = 0.005). Conversely, the proportion of individuals carrying at least one HLA-DRB1 allele encoding the D70+ was significantly decreased (p(c) = 0.004, OR = 0.4, 95% CI 0.2-0.7) among RA patients compared with controls. In conclusion, the SE is associated with RA in Mexican Mestizos as well as with the highest titers of RF.

Polymorphism of the human retinoid X receptor beta and linkage disequilibrium with HLA-DPB1.

The human retinoid X receptor beta (RXRB) gene is localized in the major histocompatibility complex (MHC) region between DPB1 and RING2. The RXRB gene sequence reported by different investigators suggests that the gene may be polymorphic. In this study, we confirmed one polymorphism by sequencing genomic DNA from four Caucasian individuals. We also developed a restriction fragment length polymorphism (RFLP) analysis to detect this specific polymorphism. Linkage analysis studies between RXRB alleles and a number of HLA markers showed significant linkage disequilibrium between RXRB*T and HLA-DPB1*0401.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity.

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Identification of most frequent HLA class I antigen specificities in Botswana: relevance for HIV vaccine design.

Since the mid-1990s, southern African countries have been experiencing an expansion of human immunodeficiency virus type 1 (HIV-1) infection caused by HIV-1 subtype C. To facilitate the design of an HLA-based HIV vaccine, we studied the distribution of the HLA class I antigen specificities in Botswana, a southern African country with a high prevalence of HIV infection. Botswana's highly efficient health care system and its central geographical location within southern Africa suggests that it might be an appropriate candidate site for future trials of an HLA-based HIV vaccine. Specificities of HLA class I genes have been investigated in DNA samples obtained from 161 persons of Botswana origin by polymerase chain reaction (PCR) with sequence-specific primers. We identified 4 HLA-A, 7 HLA-B, and 5 HLA-C specificities that were observed at high frequencies in the Botswana population: A30, A02, A23, A68, B58, B72, B42, B8, B18, B44, B45, Cw7, Cw2, Cw17, Cw6, and Cw4. HLA-A30, A02, A23, A68, B58, Cw2, Cw4, Cw6, Cw7, and Cw17 were observed at frequencies of more than 10%. The frequency of HLA-A30 was 27.3%. HLA-B58 (17.9%) was the most frequent generic HLA-B type. Other frequent antigen specificities detected for the HLA-B were B72 (9.6%), B42 (9.3%), B8 (7.4%), B18 (7.4%), B44 (7.4%), and B45 (6.4%). Analysis of haplotype frequencies revealed that haplotypes HLA-A30/HLA-B58 (6.7%), A30/B42 (6.1%), A30/B8 (4.1%), A30/B45 (3.2%), and A23/B58 (2.5%) were the most frequent among two-locus haplotypes. The comparison of HIV-positive patients and noninfected controls for HLA class I specificities confirmed the previously described association of A2/A6802 supertype with resistance to HIV. Our study suggested an increased resistance to HIV infection associated with A68 rather than A2. We also found that the generic HLA-B58 type was associated with increased susceptibility to HIV infection. Our findings suggest that the design of an HLA-based HIV vaccine that includes multiple CTL epitopes restricted by identified common HLA class I specificities might target up to 97.5% of the population in Botswana. The results of this study extend the HLA map to a southern African country that has high rates of HIV and also provide a database for the design of an HLA-based HIV vaccine.

Identification of phylogenetic footprints in primate tumor necrosis factor-alpha promoters.

The human tumor necrosis factor-alpha (TNF-alpha) gene encodes a pleiotropic cytokine that plays a critical role in basic immunologic processes. To investigate the TNF-alpha regulatory region in the primate lineage, we isolated TNF-alpha promoters from representative great apes, Old World monkeys, and New World monkeys. We demonstrate that there is a nonuniform distribution of fixed human differences in the TNF-alpha promoter. We define a "fixed human difference" as a site that is not polymorphic in humans, but which differs in at least one of the seven primate sequences examined. Furthermore, we identify two human TNF-alpha promoter single nucleotide polymorphisms that are putative ancestral polymorphisms, because each of the human polymorphic nucleotides was found at the identical site in at least one of the other primate sequences. Strikingly, the largest conserved region among the primate species, a 69-nt "phylogenetic footprint," corresponds to a region of the human TNF-alpha promoter that forms the transcriptionally active nucleoprotein-DNA complex, essential for gene regulation. By contrast, other regions of the TNF-alpha promoter, which exhibit a high density of variable sites, are nonessential for gene expression, indicating that distinct TNF-alpha promoter regions have been subjected to different evolutionary constraints depending on their function. TNF-alpha is the first case in which a promoter region dissected by functional analyses can be correlated with nucleotide polymorphism and variability in primate lineages. The results suggest that patterns of polymorphism and divergence are likely to be useful in identifying candidate regions important for gene regulation in other immune-response genes.

In situ analysis of B7-2 costimulatory, major histocompatibility complex class II, and adhesion molecule expression in schistosomal egg granulomas.

Immunopathological damage in schistosomiasis consist of egg granuloma formation, which is a hypersensitivity reaction mediated by antigen-specific CD4+ T helper (Th) lymphocytes. Th cells are dependent on accessory cell-bound co-stimulatory signals for activation. The B7 molecules provide critical costimulation, as in their absence T cells are rendered unresponsive. We investigated the kinetics of B7-2 molecule expression in schistosomal egg granulomas by immunocytochemical analysis in situ. We also monitored major histocompatibility complex (MHC) class II, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression, as well as the presence of macrophages, T cells, and B cells. B7-2 expression was elevated in cells of the large granulomas of the acute (6-1/2 week) infection, and sharply declined thereafter. MHC class II expression was similarly elevated in the acute granulomas, but in contrast to B7-2, remained relatively constant throughout 12 1/2 weeks of infection. Moreover, ICAM-1 and VCAM-1 were expressed in acute and chronic granulomas. However, whereas ICAM-1 was constitutively expressed in normal liver, VCAM-1 was dramatically induced in the livers upon onset of disease. The findings suggest that the acute granulomas are rich in accessory cells with overall phenotypic configurations that support Th-cell activation, although at subsequent times, granulomas contain increasing numbers of B7-poor accessory cells capable of inducing Th-cell unresponsiveness. Thus, cellular interactions in early granulomas may be able to amplify egg-induced immunopathology, whereas interactions taking place in succeeding granulomas appear to precipitate its down-regulation.

Recombinant IL-10 and IL-10/Fc treatment down-regulate egg antigen-specific delayed hypersensitivity reactions and egg granuloma formation in schistosomiasis.

In infection with the helminth Schistosoma mansoni, parasite eggs elicit a Th cell-mediated hepatic granulomatous inflammation that leads to scarring, portal hypertension, hemorrhage, and death. On the other hand, the cytokine IL-10 has been shown to decrease egg Ag-specific Th cell responses by down-regulating MHC class II as well as B7 costimulatory molecule expression on accessory cells. We now test the effect of IL-10 in vivo on models of egg Ag-specific hypersensitivity as well as on the natural disease. Systemic administration of IL-10 significantly inhibited delayed-type hypersensitivity reactions to egg Ag as well as primary and secondary granuloma formation to eggs embolized in the lung. However, significant inhibition of hepatic granuloma formation associated with the natural infection required the use of an IL-10/Fc fusion protein with a prolonged in vivo half-life. Lymph node cells from IL-10/Fc-treated mice produced less IL-2 and IFN-gamma, and more IL-4 and IL-10 than control cells, suggesting that reduced egg granuloma formation resulted primarily from down-regulation of Th1 responses. These results indicate that suitable administration of exogenous IL-10 can be effective in ameliorating immunopathologic damage associated with schistosomiasis.

In situ analysis of cytokine responses in experimental murine schistosomiasis.

BACKGROUND: Infection with schistosomiasis results in CD4+ T helper (Th) cell-dependent granulomatous inflammation around parasite eggs. T cell-derived cytokines play a critical role in the induction and subsequent down-modulation of the granulomas. These cytokine responses have been previously examined in lymphoid cell supernatants or in tissue homogenates but have not been examined directly in the local microenvironment of the lesions or in the reacting lymphoid tissues. EXPERIMENTAL DESIGN: With the use of specific mAb, the cytokines IL-2, IFN gamma, IL-4, and IL-10 were investigated by direct immunocytochemical analysis in situ in hepatic egg granulomas and lymphoid organs from acutely and chronically infected mice. Cytokine expression in situ was compared with cytokine production during a secondary response in vitro. RESULTS: All cytokines examined were detected in various amounts in both the hepatic egg granulomas as well as in mesenteric lymph nodes and spleen. The majority of cells expressing the cytokines was found in lymph nodes, and very few were found in granulomas. Relatively small numbers of granulomas contained most of the cytokine-expressing cells, which tended to localize in their periphery. Granulomas and lymphoid organs in the acute disease contained significantly more cytokine-expressing cells in comparison with those from the chronic disease. This observation correlated well with cytokine production in vitro. CONCLUSIONS: Direct immunocytochemical examination in situ was used to detect and measure cytokine-producing cells in hepatic egg granulomas and reacting lymphoid organs of acute and chronic experimental murine schistosomiasis. Observed cytokine patterns suggest that granulomas contain T lymphocytes of both the Th-1 and Th-2 types and that cytokine production occurs during a limited time in the early granuloma. The immunocytochemical technique affords a direct appraisal of amount, dynamics, and localization of cytokine-producing cells in the unperturbed local environment.

Accessory cell signals regulate Th-cell responses: from basic immunology to a model of helminthic disease.

Schistosome helminths inflict serious tissue damage by eliciting T helper (Th)-cell-mediated granulomatous inflammation around parasite eggs. The egg granulomas are large in acute disease and smaller in chronic disease. To explain this downregulation in chronic disease, Miguel Stadecker and Pedro Flores Villanueva describe a mechanism whereby CD4+ Th1-type lymphocytes, which are associated with the initial vigorous granuloma formation, are rendered anergic to subsequent antigenic stimulation. This results in the reduction of granuloma size and in the dominance of Th2-type lymphocyte responses.

Regulation of T helper cell responses in experimental murine schistosomiasis by IL-10. Effect on expression of B7 and B7-2 costimulatory molecules by macrophages.

Granulomatous inflammation in schistosomiasis is a manifestation of cell-mediated hypersensitivity to parasite egg Ags that is predictably reduced in size over the course of the disease. This down-regulation may reflect a state of anergy in the T cells mediating granuloma formation after interaction with accessory cells incapable of providing full stimulation. The present studies were conducted to investigate this mechanism at the molecular level. We found that granuloma macrophages (GM) strongly inhibit the ability of splenic APC to stimulate egg Ag-specific Th1 responses. This property was shown to be dependent on their secretion of IL-10. Moreover, activated GM in culture were found to express little or no costimulatory Ags B7 or B7-2. However, when their autocrine secretion of IL-10 was neutralized with specific mAb, GM displayed an up-regulation of costimulatory molecules as well as of MHC class II Ags. Most importantly, GM cultured in the presence of anti-IL-10 mAb, acquired the ability to stimulate egg Ag-specific T cells. By independently blocking each of the induced costimulatory Ags, it appeared that B7-2 molecules provided stronger costimulation than B7. In separate experiments, culture supernatants from GM exerted a powerful inhibition of costimulatory Ag expression on Con-A-stimulated peritoneal exudate cells in vivo, which could similarly be attributed to IL-10. Our results demonstrate that IL-10 can play a critical role in the generation of accessory cells that, by virtue of down-regulation of costimulatory molecules, may be capable of inducing anergy in T cells mediating the vigorous granulomatous response of acute stage schistosomiasis. Our studies lend support to the contention that a state of unresponsiveness in pathogenic T cells may precipitate the down-regulation of granuloma formation and provide a molecular basis for the underlying mechanisms.

Macrophages from schistosomal egg granulomas induce unresponsiveness in specific cloned Th-1 lymphocytes in vitro and down-regulate schistosomal granulomatous disease in vivo.

We previously have proposed that accessory cells from individuals infected with schistosomiasis induce unresponsiveness in specific Th-1 lymphocytes resulting in the immunologic down-regulation of egg-induced granulomatous inflammation characteristically seen in this disease. We now show that macrophages isolated from schistosomal egg granulomas (GM) fail to serve as stimulatory APC for cloned, murine schistosomal egg Ag (SEA)-specific, CD4+, Th-1-type lymphocytes, while being able to stimulate Th-2-type responses. Instead, GM render Th-1 cells unresponsive to restimulation. Based on these findings, we tested two approaches to down-regulate the granulomatous inflammation associated with a schistosomal challenge infection. First, active ectopic granuloma induction in response to Schistosoma mansoni eggs, but not to Ascaris lumbricoides eggs, resulted in a significant reduction of granulomatous disease in vivo, as well as in the inhibition of specific proliferation of mesenteric lymph node cells in vitro. Second, passive administration of purified GM, but not of normal peritoneal cells (PC), resulted, similarly, in significant reduction of granuloma size in vivo and specific lympho-proliferation in vitro. Moreover, cytokine analysis of supernatants from stimulated lymph node cells disclosed that the Th-1-derived cytokine IL-2 decreased to undetectable levels, at the same time as the Th-2-derived cytokines IL-4 and IL-10 increased, in animals receiving GM, in contrast to those receiving PC or no cells. These findings are consistent with the interpretation that accessory cells such as GM induce unresponsiveness in specific Th-1 cells that, in turn, results in the down-regulation of granulomatous schistosomal disease and its in vitro correlates.

Role of IL-10 on antigen-presenting cell function for schistosomal egg-specific monoclonal T helper cell responses in vitro and in vivo.

Egg Ag-stimulated lymphoid cell culture supernatants from schistosome-infected mice significantly inhibited Ag-specific, MHC-restricted proliferative responses of cloned schistosomal egg Ag-specific CD4+, Th-1-type lymphocytes. The inhibitory molecule in the supernatants was found to be the cytokine IL-10. Maximal IL-10 was produced by cells from mice carrying 8-wk schistosome infections, and none by cells from normal mice. IL-10 exerted its biologic activity on APC, and not directly on the cloned lymphocytes, resulting in the inhibition of Th-1 lymphocyte proliferation, whereas Th-2 responses were not affected. Most significantly, IL-10 also affected Th-1 clone activity in vivo, as measured by the inhibition of schistosomal egg Ag-specific local delayed-type hypersensitivity reactions. IL-10 produced in schistosome-infected individuals may play a role in the generation of APC, such as macrophages in schistosomal egg granulomas, unable to efficiently stimulate, but capable of inducing a state of anergy/unresponsiveness in Th-1 lymphocytes. We believe that this state of Th-1 cell anergy translates into the down-regulation of granulomatous hypersensitivity (immunomodulation) characteristically observed in the evolving schistosomal disease.