William R. Berrington

Mycobacterium tuberculosis, macrophages, and the innate immune response: does common variation matter?

Summary:  Despite the discovery of the tuberculosis (TB) bacillus over 100 years ago and the availability of effective drugs for over 50 years, there remain a number of formidable challenges for controlling Mycobacterium tuberculosis (MTb). Understanding the genetic and immunologic factors that influence human susceptibility could lead to novel insights for vaccine development as well as diagnostic advances to target treatment to those who are at risk for developing active disease. Although a series of studies over the past 50 years suggests that host genetics influences resistance to TB, a comprehensive understanding of which genes and variants are associated with susceptibility is only partially understood. In this article, we review recent advances in our understanding of human variation of the immune system and its effects on macrophage function and influence on MTb susceptibility. We emphasize recent discoveries in human genetic studies and correlate these findings with efforts to understand how these variants alter the molecular and cellular functions that regulate the macrophage response to MTb.

Altered Inflammatory Responses in TLR5-Deficient Mice Infected with Legionella pneumophila

Legionella pneumophila (Lp), an important cause of morbidity and mortality from pneumonia, infects alveolar macrophages (AMs) and is recognized by several TLRs as well as Birc1e (NAIP5) and IL-1 converting enzyme-protease activating factor. We examined the role of TLR5 during the murine response to aerosolized Lp infection. At 4 h after infection, Tlr5−/− mice had lower numbers of polymorphonuclear neutrophils (PMNs) in their broncho-alveolar lavage fluid in comparison to wild-type (WT) mice. At 24 and 72 h, the PMN recruitment was similar. WT mice infected with a flagellin-deficient strain (LpFlaA−) also showed an impaired early PMN response at 4 h compared with those infected with the WT strain. There was no consistent difference in bacterial counts at any of the time points when comparing the Tlr5−/− and WT mice. However, at 6 days after infection, the Tlr5−/− mice had increased leukocytic infiltrates in the alveolar and peribronchial interstitial spaces that were consistent with organizing pneumonia. We also examined the role of TLR5 during macrophage infection. In contrast to bone marrow-derived macrophages, AMs secreted TNF-α after stimulation with purified flagellin. In addition, WT, but not Tlr5−/−, AMs produced TNF-α after stimulation with Lp. Live LpFlaA− did not induce TNF-α secretion in AM. These results suggested that AMs recognize Lp flagellin and that a majority of the Lp-induced TNF-α response is TLR5-mediated. Thus, TLR5 mediates recognition of Lp in AMs and performs a distinct role during the in vivo pulmonary immune response through regulation of early PMN recruitment and subsequent later development of pneumonia.

Leprosy and the Human Genome

SUMMARY Despite the availability of effective treatment for several decades, leprosy remains an important medical problem in many regions of the world. Infection with Mycobacterium leprae can produce paucibacillary disease, characterized by well-formed granulomas and a Th1 T-cell response, or multibacillary disease, characterized by poorly organized cellular infiltrates and Th2 cytokines. These diametric immune responses confer states of relative resistance or susceptibility to leprosy, respectively, and have well-defined clinical manifestations. As a result, leprosy provides a unique opportunity to dissect the genetic basis of human in vivo immunity. A series of studies over the past 40 years suggests that host genes influence the risk of leprosy acquisition and the predilection for different clinical forms of the disease. However, a comprehensive, cellular, and molecular view of the genes and variants involved is still being assembled. In this article, we review several decades of human genetic studies of leprosy, including a number of recent investigations. We emphasize genetic analyses that are validated by the replication of the same phenotype in independent studies or supported by functional experiments demonstrating biological mechanisms of action for specific polymorphisms. Identifying and functionally exploring the genetic and immunological factors that underlie human susceptibility to leprosy have yielded important insights into M. leprae pathogenesis and are likely to advance our understanding of the immune response to other pathogenic mycobacteria. This knowledge may inform new treatment or vaccine strategies for leprosy or tuberculosis.

Common Polymorphisms in the NOD2 Gene Region Are Associated with Leprosy and Its Reactive States

BACKGROUND Because of its wide spectrum of clinical manifestations and its well-defined immunological complications, leprosy is a useful disease for studying genetic regulation of the host response to infection. We hypothesized that polymorphisms in the nucleotide-binding oligomerization domain containing 2 (NOD2) gene, for a cytosolic receptor known to detect mycobacteria, are associated with susceptibility to leprosy and its clinical outcomes. METHODS We used a case-control study design with 933 patients in Nepal. Our study included 240 patients with type 1 (reversal) reactions and 124 patients with type 2 (erythema nodosum leprosum) reactions. We compared the frequencies of 32 common polymorphisms in the NOD2 gene region between patients with the different clinical types of leprosy as well as between the patients and 101 control participants without leprosy. RESULTS Four polymorphisms were associated with susceptibility to leprosy when comparing allele frequencies, and 8 were associated when comparing genotype frequencies with a dominant model. Five polymorphisms were associated with protection from reversal reaction in an allelic analysis, and 7 were associated with reversal reaction with a dominant model. Four polymorphisms were associated with increased susceptibility to erythema nodosum leprosum in an allelic analysis, whereas 7 of 32 polymorphisms were associated with a dominant model. CONCLUSION These data suggest that NOD2 genetic variants are associated with susceptibility to leprosy and the development of leprosy reactive states.

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

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.

Clinical Correlates of Herpes Simplex Virus Viremia among Hospitalized Adults

BACKGROUND Quantification of herpes simplex virus (HSV) DNA in the peripheral blood is often used to evaluate patients suspected of having disseminated HSV infection. Few studies have examined the clinical correlates of HSV viremia among adults. METHODS We conducted a retrospective analysis of blood samples sent to a molecular virology reference laboratory at a university hospital for quantification of HSV DNA from October 2001 through June 2006. Medical records of patients with detectable HSV DNA were reviewed to abstract relevant clinical characteristics. RESULTS HSV DNA was detected in 38 (4%) of 951 samples from 29 patients, 19 of whom (66%) were >16 years old. Detailed medical records were available for review from 13 (68%) of 19 adult patients. Of the 10 patients whose HSV infection was typed, 6 (60%) had HSV-2, 3 (30%) had HSV-1, and 1 (10%) had evidence of HSV-1 and HSV-2 coinfection. All patients with viremia were treated with antiviral medications. The most common clinical findings were hepatitis (62%), fever (54%), central nervous system alterations (46%), skin lesions (38%), abdominal pain (31%), and sepsis (31%). Respiratory failure (23%) was uncommon. Patients with HSV viremia were observed to have a high mortality rate (6 of 10 immunocompromised and 1 of 3 immunocompetent individuals). CONCLUSIONS HSV viremia may be associated with a variety of signs and symptoms of morbidity in immunocompetent and immunocompromised hospitalized adults and is associated with high rates of mortality, although causality can be determined only by additional studies.

Association of TNF, MBL, and VDR polymorphisms with leprosy phenotypes

Although genetic variants in tumor necrosis factor (TNF), mannose binding lectin (MBL), and the vitamin D receptor (VDR) have been associated with leprosy clinical outcomes, these findings have not been extensively validated. We used a case-control study design with 933 patients in Nepal, which included 240 patients with type I reversal reaction (RR), and 124 patients with erythema nodosum leprosum (ENL) reactions. We compared genotype frequencies in 933 cases and 101 controls of seven polymorphisms, including a promoter region variant in TNF (G -308A), three polymorphisms in MBL (C154T, G161A and G170A), and three variants in VDR (FokI, BsmI, and TaqI). We observed an association between TNF -308A and protection from leprosy with an odds ratio of 0.52 (95% confidence interval = 0.29-0.95, p = 0.016). MBL polymorphism G161A was associated with protection from lepromatous leprosy (odds ratio = 0.33, 95% confidence interval = 0.12-0.85, p = 0.010). VDR polymorphisms were not associated with leprosy phenotypes. These results confirm previous findings of an association of TNF -308A with protection from leprosy and MBL polymorphisms with protection from lepromatous leprosy. The statistical significance was modest and will require further study for conclusive validation.

The Role of NOD2 in Murine and Human Melioidosis

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low-dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared with wild-type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis, and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole-blood stimulation with the NOD2 ligand, muramyl dipeptide, or B. pseudomallei. To our knowledge, these findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.

Genetic Variation in Toll-Interacting Protein Is Associated With Leprosy Susceptibility and Cutaneous Expression of Interleukin 1 Receptor Antagonist

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 individuals susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

Differential Dermal Expression of CCL17 and CCL18 in Tuberculoid and Lepromatous Leprosy

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.