Analysis of Host-Pathogen Interactions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Using intravital 2-photon microscopy methods developed in the LBS, we have imaged immune effector cell behavior during infectious processes. We previously described lymphoid and myeloid cell dynamics in mycobacteria-induced granulomas and showed that only a small fraction of the antigen-specific T cells undergo migration arrest at any time. This small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma. These findings provided entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. The finding that only a small fraction of the cells being imaged in a tissue are actually engaged in effector function at any time and that these have a dynamic behavior distinct from the bulk of the imaged cells raises critical questions about many existing and ongoing studies in other laboratories using intravital 2-photon imaging, in which the bulk or average behavior of clonally-related cells is taken as representative of the functional population. In other studies, we examined how the structure of lymph nodes is organized to limit access of invading organisms to the blood stream. These studies showed a crucial role of CD169+ subcapsular sinus macrophages in responding to lymph borne pathogens by producing the cytokine IL-18, which together with IL-12 or type 1 interferon, resulted in rapid cytokine-induced IFNgamma production by a diverse set of lymphocytes (memory CD8 T cells, gamma-delta T cells, NK cells, and NKT cells). Remarkably, these various lymphocytes population show constrained localization to the regions near to the subcapsular sinus-lining CD169+ macrophages, revealing a very specific tissue micro-anatomy that supports robust innate responses to incoming pathogens to present their dissemination. We have also studied the interaction of microbes with the gut, in particular, examining how potential pathobionts are constrained to exist in a commensal-like homeostatic state with the host through the action of a combination of innate lymphocytes (ILC) and T cells. Using our new static imaging tools, we have been able to quantify the extent of cytokine signaling of epithelial cells in the small bowel resulting from IL-23 induction of IL-22 from ILC3 and to determine that these ILC operate in conjunction with Th17and Treg to shape the normal commensal microbiota. ILC3s and adaptive CD4 T cells participate sequentially to establish the mature state of non-inflammatory commensalism with certain bacteria. Strikingly, the ILC3 and CD4+ T cells use different mechanisms in regulating bacterial numbers. This raises doubts about the usual assumption that ILC subsets and CD4 effector subsets share effector mechanisms but differ only in whether an antigen-specific receptor is used to drive activation. Further, we have discovered that one of the reasons evolution employs ILCs early then switches to adaptive T cell immunity over time is to prevent persistent IL-22 production by ILC3s, which causes epithelial cells to decrease lipid transporter expression, resulting in loss of adipose tissue and blood lipid dysregulation in the host. Our imaging methods have also been employed to discover the site of IL-25 action and to detail the proliferation and migration of activated ILC2 from gut to peripheral organs where they mediate anti-helminth protective immunity. These studies show that in contrast to the usual assumption that ILC are tissue resident effector cells, they have a similar behavior to adaptive T cells in that they are activated, proliferate, and differentiate in one tissue (in the case of iILCs, the small bowel lamina propria), the migrate into lymph and then the blood circulation in an S1P-dependent manner to exit at a distant tissue to mediate protective function. The molecular mechanisms employed by the ILC2s to leave the intestine is thus the same as that employed by adaptive T cells to exit the lymph node after activation, involving S1PR sensing of an S1P gradient between the tissue and blood. The drug FTY720 prevents ILC2s from exiting the intestine after IL-25 activation, preventing them from mediating effective protection against helminth infection involving the lungs, to which the activated ILC2s usually travel. Most recently, we have returned to the study of lethal influenza infection. Using data from our previous systems-level analysis, we examined whether tissue based RNA expression data could inform a blood signature that would allow prediction of lethal infection at an early stage. We could validate the specificity of the lethal vs. influenza infection pan signature and predict lethality at one LD50 in infected mice, especially with the addition of markers for activated T cells. These data suggest that stochastic variation in antigen specific immune repertoire among the infected animals can determine the difference between life and death, with important implications for understanding many lethal human viral infections. We are also using this model and imaging tools as the substrate for our screening of drugs for treatment efficacy late in disease and for tests to follow using mouse SARS-Cov2 models (see AI001292-02). Collaborations are also in progress to use our imaging tools to (i) analyze the control of the generation and functioning of stem-like memory CD8 T cells, a population that plays a critical role in the efficacy of checkpoint immunotherapy and (ii) study the very early events in the innate immune response to viral infection, initially using VSV as a model system and MAVS-deficient mice as compared to intact animals to study the role of this sensor system in type 1 interferon production in constraining viral spread. In connection with the several viral infection models we are employing in our work, we have optimized two different approaches to multiplex imaging. For the lung infection studies with influenza (and eventually SARS-Cov2) we have created a pipeline for examining sections of entire lung lobes with antibodies that are directed to innate and adaptive immune subsets, endothelial and epithelial cells, extracellular stromal elements and products of specialized alveolar epithelial cells, along with signaling molecules like pSTAT1. For the VSV work, we have expanded our staining panels for studies in lymph nodes.