Jeff F. Miller

Type I Interferon Production Enhances Susceptibility to Listeria monocytogenes Infection

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes–induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.

Involvement of receptor-interacting protein 2 in innate and adaptive immune responses

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-κB, whereas dominant negative Rip2 inhibited NF-κB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-γ production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.

Compartmentalization of Bacterial Antigens: Differential Effects on Priming of CD8 T Cells and Protective Immunity

Bacterial pathogens synthesize numerous proteins that are either secreted or localized within bacterial cells. To address the impact of antigen compartmentalization on T cell immunity, we constructed recombinant Listeria monocytogenes that express a model CD8T cell epitope as a secreted or nonsecreted fusion protein. Both forms of the antigen, either secreted into the host cell cytoplasm or retained within bacterial cells, efficiently prime CD8 T cell responses. However, epitope-specific CD8 T cells confer protection only against bacteria secreting the antigen but not against the bacteria expressing the nonsecreted form of the same antigen. This dichotomy as a result of antigen compartmentalization suggests that bacterial antigens are presented by multiple MHC class I pathways to prime CD8 T cells, but only the endogenous pathway provides target antigens for CD8 T cell-mediated protective immunity.

Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction

Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.

Roles for motility in bacterial–host interactions

The ability to move in a directed manner may confer distinct advantages upon host‐adapted prokaryotes. Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts and access optimal colonization sites within them, and dispersal in the environment during the course of transmission. The costs of motility also may be significant. These include the metabolic burden of synthesizing flagellar components, the energetic expense of fuelling flagellar motors and the presentation of polymeric and highly antigenic targets to the immune system. It is therefore not surprising that synthesis of the motility apparatus is usually subject to strict control. Studies of a variety of bacterial–host interactions demonstrate roles for motility, and its regulation, at points throughout the infectious cycle.

Propionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne

The human skin microbiome plays important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that while the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may play roles in determining virulence properties of P. acnes strains and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain level analysis of the human microbiome to define the role of commensals in health and disease.

Original ArticlePropionibacterium acnes Strain Populations in the Human Skin Microbiome Associated with Acne

The human skin microbiome plays important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that while the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may play roles in determining virulence properties of P. acnes strains and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain level analysis of the human microbiome to define the role of commensals in health and disease.

Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome

Mice with a targeted disruption of the gene encoding a lymphoid-expressed orphan G protein-coupled receptor, G2A, demonstrate a normal pattern of T and B lineage differentiation through young adulthood. As G2A-deficient animals age, they develop secondary lymphoid organ enlargement associated with abnormal expansion of both T and B lymphocytes. Older G2A-deficient mice (>1 year) develop a slowly progressive wasting syndrome, characterized by lymphocytic infiltration into various tissues, glomerular immune complex deposition, and anti-nuclear autoantibodies. G2A-deficient T cells are hyperresponsive to TCR stimulation, exhibiting enhanced proliferation and a lower threshold for activation. Our findings demonstrate that G2A plays a critical role in controlling peripheral lymphocyte homeostasis and that its ablation results in the development of a novel, late-onset autoimmune syndrome.

The TLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: Relation to central nervous system antitumor immunity

Immunotherapy represents an appealing option to specifically target CNS tumors using the immune system. In this report, we tested whether adjunctive treatment with the TLR-7 agonist imiquimod could augment antitumor immune responsiveness in CNS tumor-bearing mice treated with human gp100 + tyrosine-related protein-2 melanoma-associated Ag peptide-pulsed dendritic cell (DC) vaccination. Treatment of mice with 5% imiquimod resulted in synergistic reduction in CNS tumor growth compared with melanoma-associated Ag-pulsed DC vaccination alone. Continuous imiquimod administration in CNS tumor-bearing mice, however, was associated with the appearance of robust innate immune cell infiltration and hemorrhage into the brain and the tumor. To understand the immunological mechanisms by which imiquimod augmented antitumor immunity, we tested whether imiquimod treatment enhanced DC function or the priming of tumor-specific CD8+ T cells in vivo. With bioluminescent, in vivo imaging, we determined that imiquimod dramatically enhanced both the persistence and trafficking of DCs into the draining lymph nodes after vaccination. We additionally demonstrated that imiquimod administration significantly increased the accumulation of tumor-specific CD8+ T cells in the spleen and draining lymph nodes after DC vaccination. The results suggest that imiquimod positively influences DC trafficking and the priming of tumor-specific CD8+ T cells. However, inflammatory responses induced in the brain by TLR signaling must also take into account the local microenvironment in the context of antitumor immunity to induce clinical benefit. Nevertheless, immunotherapeutic targeting of malignant CNS tumors may be enhanced by the administration of the innate immune response modifier imiquimod.

A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens.

The Bordetella BvgAS signal‐transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg+ phase, characterized by the expression of adhesins and toxins, and the Bvg− phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. In B. bronchiseptica, the Bvg+ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg− phase is required for growth under nutrient‐limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg‐intermediate (Bvgi) phase. The mutation conferring this phenotype, designated bvgS‐I1, results in a threonine‐to‐methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg+‐phase‐locked parent, the Bvgi mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg+‐phase factors and has gained the ability to express factors unique to the Bvgi phase. Although identified by mutation, this work indicates that the Bvgi phase is expressed by wild‐type B. bronchiseptica in response to certain (semi‐modulating) environmental conditions. The identification of Bvgi‐specific antigens suggests the existence of a new class of Bvg‐regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.