Chougnet Lab

Regulatory T cells (Treg), a subset of CD4⁺ T cells, are required both to establish and sustain immunological self-tolerance. We have shown during chronic infections, such as HIV infection, that Treg accumulate and they preserve their functionality (JI, 2005; Blood, 2006; J Virol, 2007). We have also shown that Treg act as double-edge sword during HIV infection, blunting immune responses (Blood, 2006), but also decreasing T cell and dendritic cell infection by HIV (Blood, 2012; Front Immunol, 2014). In addition, we study the effect of highly active antiretroviral therapy on Treg function and homeostasis (PLoSOne, 2011, and 2013).

Our lab also studies Treg biology in other settings. Treg develop very early during the ontogeny of the human infant immune system and their disappearance and/or dysfunction is likely to play a role in the debilitating inflammatory diseases that premature neonates developed, such as Broncho-Pulmonary Dysplasia or Necrotizing Enterocolitis. Importantly, over 50% of very preterm births have an antecedent infection associated with chorioamnionitis or premature rupture of membranes. In collaboration with Drs. Alan Jobe and Suhas Kallapur in the Division of Neonatology, we are actively pursuing this line of research focusing on the effect of chorioamnionitis on Treg homeostasis and function, either in nonhuman primate experimental models (JI, 2013) or in human cohorts (Hum Immunol, 2014). We also collaborate with Dr. Alex Miethke in the Division of Gastroenterology and Nutrition to study the role of Treg in biliary atresia (J Hepatol, 2010, Hepatology, 2012).

We have shown that Treg homeostasis is changed during aging, leading to increased survival and accumulation in lymphoid organs (JI, 2008; JI, 2011; Front Immunol, 2013). We are now deciphering the underlying mechanisms as Treg accumulation is an important contributing factor to immunosenescence.

To better study human Treg function, we have developed advanced phenotypic strategies (Cytometry A, 2010) and assays that probe Treg suppressive action on dendritic cells and their capacity to activate conventional T cells, using imaging flow cytometry (Figure 1 and Front Immunol, 2014).

Funding:

• NIH R01 AG033057 (co-PIs: C. Chougnet/D. Hildeman) Homeostasis and function of regulatory T cells in aging.

• NIH U01HL101800 (co-PIs: A. Jobe/C. Chougnet) Biomarkers of Immunologic Function and Preterm Respiratory Outcomes.

• NIH R01HD078127 (co-PIs: L. Muglia/C. Chougnet) Maternal temperament, stress, and inflammation in preterm birth.

• Burrough-Wellcome fund (PI: C. Chougnet) Host-microbe cross talk and pregnancy outcomes.
• Cincinnati Children's RIP (PI: C. Chougnet) Mechanisms underlying High Density Lipoprotein-mediated survival of human regulatory T cells.

Analysis of human Regulatory T cell (Treg) function.

We developed assays that probe Treg suppressive activity on dendritic cell (DC)-mediated l stimulation of conventional T cells (Tcon). Treg were added or not to DC and Tcon cultures at a physiological ratio of 1:10:1(DC:Tcon:Treg) ratio. (A) 3 populations can be visualized by imaging flow cytometry: (1) isolated DC (HLA-DR⁺ cells), (2) isolated Tcon (CellTrace^TM-Violet⁺ cells) and (3) DC:Tcon aggregates (CellTrace^TM-Violet⁺HLA^-DR⁺ population). (B) Actin polymerization at the immunological synapse forming between DC and Tcon. (C) Treg control different aspects of DC-Tcon activation, decreasing the percentage of DC:Tcon aggregates in the cellular population, the percentage of activated Tcon (CD134⁺), the percentage of activated DC (CD83⁺) in aggregates and actin polymerization at the immunological synapse.

Over 50% of very preterm births have an antecedent infection associated with chorioamnionitis or premature rupture of membranes. Pathological investigations suggest an initial focal choriodecidual colonization by lower genital tract organisms (including Ureaplasma species, commonly associated with preterm birth) followed by dissemination into the amniotic fluid, and induction of preterm labor. Concurrently, the fetus is exposed to bacteria, bacterial products (such as LPS) and/or inflammatory cytokines, causing fetal inflammatory response syndrome (FIRS). However, the precise mechanisms of how microorganisms trigger inflammatory changes (in the placenta and fetus) remain poorly defined.

We are actively pursuing this line of research, principally using nonhuman primate models of experimental chorioamnionitis to characterize fetal (JI, 2013) or placental inflammation (Biol Reproduction, 2014).

Collaborators: This project is conducted in collaboration with Alan Jobe, MD, PhD, Suhas Kallapur, MD, and Louis Muglia, MD, PhD, in the Perinatal Institute, and Lisa Miller, PhD and John Capitanio PhD at the California National Primate Research Center in Davis, CA.

Funding:

• NIH U01HL101800 (co-PIs: A. Jobe/C. Chougnet) Biomarkers of Immunologic Function and Preterm Respiratory Outcomes.

• NIH 1R01HD078127 (co-PIs: L. Muglia/C. Chougnet) Maternal temperament, stress, and inflammation in preterm birth.
• Burrough-Wellcome (PI: C. Chougnet) Host-microbe cross talk and pregnancy outcomes.

Intra-amniotic IL-1β transiently decreases regulatory T cell (Treg) frequency but increases the frequency of IL-17+ cells in lymphoid organs.

Human recombinant IL-1β, or saline, was injected intra-amniotically to pregnant rhesus macaques at ~80% gestation. At 24h or 48h post-injection, fetuses (n=4-6/group) were delivered by C-section and immune responses were analyzed in tissues (spleen and lymph nodes) by flow cytometry. Data from the spleen are shown. (A) Mean (SE) Treg frequency (defined as percentage of FOXP3⁺ cells in the gated CD3⁺CD4⁺ population). (B) Mean percentages of IL-17A⁺ cells in gated CD3⁺CD4⁺ cells were measured after short in vitro PMA and ionomycin stimulation. Significant p values (Mann-Whitney tests) or trends are shown. From Kallapur et al, JI, 2013.

Aging has a profound negative impact on the capacity of the immune system to develop efficient effector responses against a vast array of antigens, leading to poor response to pathogens or vaccines. Importantly, aging affects all arms of the immune system, but most of the research has so far focused on the adaptive immune response.

We have shown that Treg homeostasis is changed during aging, leading to increased survival and accumulation in lymphoid organs (JI, 2008; JI, 2011; Front Immunol, 2013). We are now deciphering the underlying mechanisms as Treg accumulation is an important contributing factor to immunosenescence.

Impaired functionality of dendritic cells (DC) is likely a significant element of this decreased response, and deciphering the underlying mechanisms has thus become an important area of investigation. In collaboration with Dr. Edith Janssen (Division of Immunobiology), we focus on this aspect of DC biology, linking age-related changes in mitochondrial functions with the impaired capacity of the aged DC to prime CD8⁺T cells to cell-associated antigens

Natural killer cells (NK cells) also have impaired maturation and function in aging. Our work, in collaboration with Dr. Kasper Hoebe suggests that the aged non-hematopoietic environment is responsible for these defects (Aging Cell, 2014). We are further defining these non-hematopoietic factors, as identifying them could open new therapeutic avenues to improve NK cell function in the elderly.

Collaborators: David Hildeman PhD, Kasper Hoebe PhD

Funding:

NIH R01 AG033057 (co-PIs: C. Chougnet/D. Hildeman) Homeostasis and function of regulatory T cells in aging.

NK cell maturation is impaired and is controlled by non-hematopoietic factors

(A): Proportion of splenic NK subsets (double negative, immature, transitional and mature) in aged and young mice. p values represent the differences between young and aged mice. *P<0.05, **P<0.01 and ***P<0.005. (B) to (C): Proportion of splenic NK subsets at 2 weeks (B) and 6 weeks (C) post bone marrow chimera in a young or aged environment. p values represent the difference between NK cells maturing in a young vs. aged environment. From Shehata et al, Aging Cell, in press.