HIF-1 in intestinal innate immune and barrier functions in context of infections with Salmonella Typhimurium

  • HIF-1 im Zusammenhang mit angeborenen Immun- und Barrierefunktionen des Darms im Kontext von Infektionen mit Salmonella Typhimurium

Robrahn, Laura Marie; van Dongen, Joost Thomas (Thesis advisor); Cramer, Thorsten (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


The hypoxia-inducible factor (HIF)-1 functions as a regulator of the cellular adaption to low oxygen levels (hypoxia) and influences the innate immune response to various pathogenic microorganisms. The heterodimeric transcription factor consists of a β and a α subunit. The α subunit is stabilized in response to hypoxia as well as different pathogenic microorganisms allowing HIF-1 transcription factor complex assembly. HIF-1 activation impacts reprogramming of cellular metabolism and immune functions. It was further shown to majorly influence the inflammatory response of phagocytes and epithelial and endothelial cells to bacterial invaders. Additionally, HIF-1 was reported to impact inflammatory conditions of the colon, such as colitis and colitis-associated cancers, albeit with conflicting results on its anti- or pro-inflammatory role. However, HIF-1 activity was linked to intestinal barrier stabilization through its influence on mucosal development and cell turnover, cell fate decisions, tight junction integrity and mucus as well as microbiota interactions. HIF-1α deficiency in the murine intestinal epithelium was further shown to increase susceptibility to Yersinia enterocolitica, while mice proved less susceptible to Clostridium difficile toxin-induced colitis upon HIF-1α stabilization. I therefore sought to address the functional importance of HIF-1 in infections with one of the major food-born, gastrointestinal pathogens worldwide, Salmonella Typhimurium. In silico analysis of published transcriptomics data of Salmonella infected murine wildtype guts displayed significantly increased HIF-1 transcription factor activity as well as target gene transcription upon infection, which could further be linked to increased non-canonical means of HIF activation. Surprisingly, the loss of Hif1a in intestinal epithelial cells in an adult murine Salmonella infection model did not alter disease progression or systemic infection, which was further confirmed with the help of a second, inducible Hif1a knockout mouse line also harboring the deletion of Hif1a in intestinal epithelial cells. The data acquired in this work did not support further HIF-2-dependent compensatory mechanisms either. Surprisingly, myeloid cell specific Hif1a deficiency lowered the systemic bacterial burden in the murine infection model. Of note, the mRNA expression of pro-inflammatory chemokine CXCL2 (Chemokine (C-X-C motif) ligand 2) in the intestinal epithelium of knockout mice was ameliorated upon infection, suggesting a potentially inflammation controlling impact of myeloid HIF-1 in intestinal infections. Since HIF-1 impacts intracellular bactericidal functions of phagocytes, intracellular bacteria "killing assays" were performed utilizing bone marrow derived macrophages from wildtype littermates and mice harboring a myeloid Hif1a knockout. Again, Hif1a deficiency seemed dispensable for intracellular Salmonella killing, however, not for Escherichia coli. Salmonella is capable of surviving in the macrophage niche, for example via the expression of a safe intracellular compartment which’s assembly is enabled via the SPI-2 T3SS (Salmonella pathogenicity island 2 Type III secretion system). Intracellular survival of Salmonella deficient for this SPI-2 T3SS, however, was comparable to the wildtype strain. An increased release of lactate dehydrogenase though argued towards Salmonella-mediated, HIF-1-independent, macrophage death instead. Systemic, more life-threatening infections with Salmonella Typhimurium are more prevalent in infants and neonates while relatively little is known about the neonate gut in comparison to the adult intestine. The impact of HIF-1 in this regard was addressed utilizing a neonate, murine Salmonella infection model which revealed increased HIF-1 stabilization as well as changes in HIF-1 target gene expression upon infection, while again, Hif1a deficiency proved dispensable for the outcome of the neonate Salmonella infection. The here acquired results clearly showed an increase of HIF-1 activity upon Salmonella infection and supported potential alterations in the mucosa and further barrier functions in response to epithelial Hif1a loss. These findings implicate that HIF-1 could still impact gastrointestinal infections with Enterobacteriacea in all age groups in a significant way, however, the well adapted pathogen Salmonella Typhimurium seems to bypass HIF-1-dependent immune functions. While the specific Salmonella virulence mechanisms responsible for this circumvention could not be identified, the pharmacological or genetical stabilization of HIF-1α could still prove beneficial to combat systemic and/or gastrointestinal Salmonella infections.