Host innate and adaptive immunity shapes the gut microbiota biogeography.

The gut microbiota has a fundamental role in the development and the maturation of the host immune system. Both innate and adaptive immune cells have critical functions in microbial pathogen containment and clearance, but the regulation of the commensal microbiome ecosystem in the gastrointestinal tract by these major immune cell populations is incompletely defined. The role of specific innate and adaptive immune cell in the regulation of the microbiota in the intestinal tract biogeographically was investigated. Dendritic cells, macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells were depleted using monoclonal antibodies and clodronate liposomes, and the microbial communities were determined by 16S rRNA gene sequencing. With specific immune cell depletion, distinct microbiota changes were observed. In general, immune cell depleted mice had higher microbiota richness and evenness at all gut anatomical sites. At each gut segment, samples from immune cell-depleted animals clustered away from the isotype/liposome control mice. This was especially dramatic for the small intestinal microbiota. Specifically, Enterobacteriaceae, Bacteroides acidifaciens, and Mucispirillum schaedleri were highly enriched in the mucosa and lumen of the small intestine in immune cell-deficient animals. Further, the mucosal microbiota had higher microbiota evenness compared with luminal microbiota at all gut segments, and the UniFrac distance between B cell depleted and isotype control mice was the largest in the duodenum followed by the ileum and colon. Taken together, the data suggest that innate and adaptive immune cells specifically contribute to the regulation of the gut microbiota's biogeographical distribution along the gastrointestinal tract, and microbiota in the duodenum mucosa are more responsive to host immune changes compared with other anatomical sites.

Analysis of the intestinal microbial community and inferred functional capacities during the host response to Pneumocystis pneumonia.

BACKGROUND: Pneumocystis pneumonia is a major cause of morbidity and mortality in patients infected with HIV/AIDS. In this study, we evaluated the intestinal microbial communities associated with the development of experimental Pneumocystis pneumonia, as there is growing evidence that the intestinal microbiota is critical for host defense against fungal pathogens. METHODS: C57BL/6 mice were infected with live Pneumocystis murina (P. murina) via intratracheal inoculation and sacrificed 7 and 14 days postinfection for microbiota analysis. In addition, we evaluated the intestinal microbiota from CD4+ T cell depleted mice infected with P. murina. RESULTS: We found that the diversity of the intestinal microbial community was significantly altered by respiratory infection with P. murina. Specifically, mice infected with P. murina had altered microbial populations, as judged by changes in diversity metrics and relative taxa abundances. We also found that CD4+ T cell depleted mice infected with P. murina exhibited significantly altered intestinal microbiota that was distinct from immunocompetent mice infected with P. murina, suggesting that loss of CD4+ T cells may also affects the intestinal microbiota in the setting of Pneumocystis pneumonia. Finally, we employed a predictive metagenomics approach to evaluate various microbial features. We found that Pneumocystis pneumonia significantly alters the intestinal microbiota's inferred functional potential for carbohydrate, energy, and xenobiotic metabolism, as well as signal transduction pathways. CONCLUSIONS: Our study provides insight into specific-microbial clades and inferred microbial functional pathways associated with Pneumocystis pneumonia. Our data also suggest a role for the gut-lung axis in host defense in the lung.

Hypothesis testing clarifies the systematics of the main Central American Chagas disease vector, Triatoma dimidiata (Latreille, 1811), across its geographic range.

The widespread and diverse Triatoma dimidiata is the kissing bug species most important for Chagas disease transmission in Central America and a secondary vector in Mexico and northern South America. Its diversity may contribute to different Chagas disease prevalence in different localities and has led to conflicting systematic hypotheses describing various populations as subspecies or cryptic species. To resolve these conflicting hypotheses, we sequenced a nuclear (internal transcribed spacer 2, ITS-2) and mitochondrial gene (cytochrome b) from an extensive sampling of T. dimidiata across its geographic range. We evaluated the congruence of ITS-2 and cyt b phylogenies and tested the support for the previously proposed subspecies (inferred from ITS-2) by: (1) overlaying the ITS-2 subspecies assignments on a cyt b tree and, (2) assessing the statistical support for a cyt b topology constrained by the subspecies hypothesis. Unconstrained phylogenies inferred from ITS-2 and cyt b are congruent and reveal three clades including two putative cryptic species in addition to T. dimidiata sensu stricto. Neither the cyt b phylogeny nor hypothesis testing support the proposed subspecies inferred from ITS-2. Additionally, the two cryptic species are supported by phylogenies inferred from mitochondrially-encoded genes cytochrome c oxidase I and NADH dehydrogenase 4. In summary, our results reveal two cryptic species. Phylogenetic relationships indicate T. dimidiata sensu stricto is not subdivided into monophyletic clades consistent with subspecies. Based on increased support by hypothesis testing, we propose an updated systematic hypothesis for T. dimidiata based on extensive taxon sampling and analysis of both mitochondrial and nuclear genes.

CD4(+) T-Cell-Independent Secondary Immune Responses to Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4(+) T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4(+) T-cells is mediated by a robust memory humoral response, CD8(+) T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8(+) T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8(+) T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4(+) T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8(+) T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8(+) T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8(+) T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ(+) CD8(+) T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8(+) T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG.

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Towards a phylogenetic approach to the composition of species complexes in the North and Central American Triatoma, vectors of Chagas disease.

Phylogenetic relationships of insect vectors of parasitic diseases are important for understanding the evolution of epidemiologically relevant traits, and may be useful in vector control. The sub-family Triatominae (Hemiptera:Reduviidae) includes ∼140 extant species arranged in five tribes comprised of 15 genera. The genus Triatoma is the most species-rich and contains important vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Triatoma species were grouped into complexes originally by morphology and more recently with the addition of information from molecular phylogenetics (the four-complex hypothesis); however, without a strict adherence to monophyly. To date, the validity of proposed species complexes has not been tested by statistical tests of topology. The goal of this study was to clarify the systematics of 19 Triatoma species from North and Central America. We inferred their evolutionary relatedness using two independent data sets: the complete nuclear internal transcribed spacer-2 ribosomal DNA (ITS-2 rDNA) and head morphometrics. In addition, we used the Shimodaira-Hasegawa statistical test of topology to assess the fit of the data to a set of competing systematic hypotheses (topologies). An unconstrained topology inferred from the ITS-2 data was compared to topologies constrained based on the four-complex hypothesis or one inferred from our morphometry results. The unconstrained topology represents a statistically significant better fit of the molecular data than either the four-complex or the morphometric topology. We propose an update to the composition of species complexes in the North and Central American Triatoma, based on a phylogeny inferred from ITS-2 as a first step towards updating the phylogeny of the complexes based on monophyly and statistical tests of topologies.

Vector blood meals and Chagas disease transmission potential, United States.

A high proportion of triatomine insects, vectors for Trypanosoma cruzi trypanosomes, collected in Arizona and California and examined using a novel assay had fed on humans. Other triatomine insects were positive for T. cruzi parasite infection, which indicates that the potential exists for vector transmission of Chagas disease in the United States.