1. Functional Microbiomics: Gut Bacteria and the Immune System
Catherine Lozupone, PhD
Assistant Professor, School of Medicine
University of Colorado Anschutz Medical Campus

2. Intestinal bacteria help to maintain immune homeostasis in the gut
Bouma et al, 2014 Nature Reviews Immunology

3. How do gut bacteria stimulate Tregs?
Mazmanian et al
Cell Host and
Microbe 2012

4. Overall Goal: To use –OMICS data to develop testable hypotheses.
16S rRNA sequencing
Genomes
Shotgun metagenomics
Microbial RNA Seq
Host RNA-Seq
Flow Cytometry
CyTOF
Metabolome
Diet surveys/ intervention
Overall Goal: To use –OMICS data to develop testable hypotheses.

5. Can we identify ZPS producers from genomes?
Neff, Rhodes et al Cell Host and Microbe.
Coyne et al. 2001

6. We predicted that
hundreds of genomes
from strains of dozens
of distinct species
Contained ZPS operons

7. Neff, Rhodes et al. 2017. Cell Host and Microbe.

8. Bacteroidales
Red:PSA producer
Black: non

9. Katie Arnolds
Preston Neff

10. Colm Collins

11. HIV infection is associated with immune and microbial dysbiosis
So why even look at the gut microbiome in HIV infection. In a healthy individuals there is important communication between the gut immune system and the gut microbiota. This crosstalk helps keep both systems at homeostatis.
But as many of you know in HIV targets activated CD4 T cells resulting in a drastic depletion and compositional alteration of immune cells and very prominently in the mucosa. This not only upsets the balance in the immune microbiome regulation but also results in a decrease of barrier integrity leading to increased microbial translocation.
Some of this aberration is remedied by antiretroviral therapy, or ART as I will refer to it in the rest of the talk, however though immune recovery is observed in the periphery on successful art, this recovery is much slower and less complete in the gut. Furthermore, we still observe sustained alterations or dysbiosis in the gut microbiome.
While our lab in partnership with Brent Palmer’s lab looks at this whole picture. My talk today will focus only on the microbiome side of this problem as understanding the microbiome composition in HIV is the pivotal first step in informing the studies of immune microbiome interactions.
the work I'm doing is instrumental in informing their data. - I'm finding changings and they are figuring out what it means
Li et al., Clin Pharmacol Ther. 2016

12. Exploring immune-modulatory properties of the microbiome with human cells in vitro
Fecal Microbiota A B C
Soluble Debris
Isolating whole
Bacterial
communities
from stool
Histodenz
Hevia et al, Scientific Rep 2015 E
HIV+
HIV-
Brent Palmer D
For our studies with primary human cell cultures, we wanted to be able to test the impact of whole bacterial communities from our stool samples, instead of using bacterial cultures of one or a few species of bacteria
Preston another post doc in the lab
10^10 bacteria per 2 g of stool
Prevotella PI
Bacteriodes TO
Whole Stool
Isolated Bacteria
Isolated Bacteria
Whole Stool
Preston Neff

13. Stimulation of PBMC with fecal microbiota
HIV- Low Risk
HIV- High Risk
HIV+ ART-
4 days
HLADR
CD38
Fecal Microbiota
Live bacteria – grown in aerobic conditions, antibiotics
PBMC

14. CD4+ and CD8+ T cells have highest activation when stimulated with stool bacteria from untreated HIV+ individuals

15. CD8 activation levels in stool bacteria positively correlate with those in blood
ci=-0.06 to 0.59
Red: HIV positive individuals
Black: HIV negative

16. Opportunistic pathogens correlate with immune activation and are increased with HIV
Terrisporobacter glycolicus
Turicibacter sanguinis
These organisms co-occur with each other and are significantly higher in HIV positive individuals.
These organisms are very rare – only making up fractions of a percent of the community at most.

17. TLR2 (LPS) and particularly TLR4 (peptidoglycan) ligands are important for immune activation

18. Summary
Integrating ‘OMICS data with experimental validation can help to yield insights into immune-modulatory bacteria, their mechanisms of action, and their role and disease.
The insights gained can be applied to many different disease systems.

19. Thanks! My lab Brent Palmer HIV study team ZPS work collaborators
Nichole Nusbacher, Jody Donnelly (Sequencing HIV samples and culturing), Moshe Rhodes (Genomic predictions), Katie Arnolds (PSA knockouts), Abigail Armstrong (HIV 16S analysis), Mike Shaffer (multi-’omics analysis), Sam Li, Cuining Liu , Keith Hazelton, Nancy Moreno-Huizar
Brent Palmer
Preston Neff (immune stimulations), Jennifer Schneider, Sharon Sen, Sam Li (immune assays and informatics), Jennifer Schneider
HIV study team
Brent Palmer, Tom Campbell Martin McCarter, Blair Fennimore, Christine Griesmer,
Suzanne Fiorillo
Janine Higgins, Tyson Marden
ZPS work collaborators
Colm Collins, Sarkis Mazmanian, Eric Martens
Funding
HIV RO1s – NIDDK
CCTSI microgrant program: These awards can be used to pay for CTRC resources and services necessary to conduct CTRC-approved protocols at University of Colorado Hospital, Children’s Hospital Colorado, and Perinatal CTRC.
NIH training grants in Computational Biosciences, HIV pathogenesis and Infectious Disease Pathogenesis