Elucidating the role of transcription factor EB in innate immune response in vivo and in vitro

Candidate: Subothan Inpanathan
Supervisor: Dr. Roberto Botelho

Abstract:

Transcription Factor E Box (TFEB) regulates adaptation of lysosomal and autophagic physiology to various stresses, including infection. For example, exposure to LPS, microbes, and phagocytosis induce nuclear translocation of TFEB to promote antimicrobial activity, not only through induction of autophagy and lysosome function, but also by expressing antimicrobial factors and immuno-modulatory cytokines, chemokines, and the metabolite itaconate. Nevertheless, microbes like Salmonella seem to actively modulate TFEB activity in a context-dependent manner. Thus, it is unclear what the functional consequences of TFEB is for Salmonella and for host cells. Moreover, the role of TFEB in innate immunity and infection resolution in a vertebrate in vivo model is unknown. Our research specifically explores the role of TFEB in host macrophages in response to E. coli and Salmonella using in vitro and in vivo models of infection.

To address the role of TFEB in macrophages, we bred flox-Tcfeb (wild-type) and LysM-Cre recombinase mice to generate myeloid-specific tcfeb-/- mice (tcfeb-/-), assessing both male and female mice. We observed no significant differences in the baseline health, breeding, and survival between the tcfeb-/- and wild-type mice. We then characterized bone-marrow derived macrophages (BMDMs) in vitro observing no differences between tcfeb-/- and wild-type BMDMs in terms of differentiation, M1 polarization, and basal lysosomal activity. However, female tcfeb-/- BMDMs, but not male BMDMs, registered higher phagocytic uptake relative to wild-type BMDMs. Regardless of this, phagocytosed E. coli survived better in BMDMs from tcfeb-/- mice relative to control cells, suggesting impaired antimicrobial activity in the absence of TFEB.

We then tested the importance of TFEB in the host innate immune response in vivo using two infection models. Lavage of intraperitoneal infection with E. coli recovered higher levels of E. coli in both supernatant (free bacteria) and associated myeloid-cell pellet from knockout mice, suggesting higher overall microbe survival. These mice also displayed decreased expression of pro-inflammatory cytokines compared to wild-type mice, suggesting that the loss of TFEB may impair immune-modulatory cytokines release. Additionally, using an oral gavage model of Salmonella infection, we observed sex-linked differences; while wild-type and tcfeb-/- female mice had similar survival in response to Salmonella, tcfeb-/- male mice were more prone to succumb to infection. However, there was no differences in the level of Salmonella present in the spleen, suggesting that dissemination of the pathogen to other organs is unaffected.

We also delineated the host-pathogen dynamics between Salmonella infection of macrophages and TFEB in vitro. We find that Salmonella manipulates TFEB in a growth-phase and time dependent manner. Stationary, SPI-2 genes expressing Salmonella restrict TFEB activity within the first hour of infection while late-log Salmonella, expressing SPI-1 genes, induces TFEB nuclear translocation in the first hour of infection. We find that stationary Salmonella restriction of TFEB relies on T3SS function and effectors PhoP, SifA and SopD2. Furthermore, we observe ectopic activation of TFEB restricts Salmonella survival in macrophages.

Taken together, the results of both the in vitro and in vivo models of infection suggest that the loss of myeloid-specific TFEB is important for the coordination of specific host responses to bacterial infection with potential differences related to biological sex and microbes. Currently, we are examining the role of TFEB in innate cell infiltration to infection sites. Ultimately, our research seeks to advance the development of novel therapeutic strategies that harness TFEB to combat infections and treat immune-related disorders.