Biology subjects

Cohn, I. S.

Publications and source records attributed to Cohn, I. S..

4 recordsLinked to original sources

bioRxiv2023

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-gamma on parasite restriction

The production of IFN-{gamma} is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. The use of single cell RNA sequencing to profile IEC during infection revealed induction of IFN-{gamma}-dependent gene signatures that was comparable between uninfected and infected cells, and IEC expression of the IFN-{gamma} receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-{gamma} demonstrated the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-{gamma} on IEC and suggest a model in which IFN-{gamma}-mediated bystander activation of uninfected enterocytes is important for control of Cryptosporidium. AUTHOR SUMMARYThe cytokine interferon-gamma (IFN-{gamma}) plays an important role in the control of intracellular infections by a wide variety of bacteria, viruses and parasites. While the impact of IFN-{gamma} on immune cells has been a major research focus, how it impacts intestinal epithelial cells remains poorly understood. Cryptosporidium parasites are an important cause of morbidity in a variety of epidemiological settings and exclusively infect intestinal epithelial cells (IEC). Recent advances in the ability to genetically modify and study Cryptosporidium in wild-type hosts provides a useful model to investigate IEC-intrinsic mechanisms of pathogen control. In this study, single cell RNA-sequencing was used to analyze the IEC response to infection and IFN-{gamma} signalling. We demonstrate broad changes in the epithelial compartment during infection that include the induction of an IEC population with robust induction of IFN-{gamma}-stimulated genes. In addition, we show that infected IEC remain responsive to IFN-{gamma} signalling, and that this cytokine causes a delayed reduction in parasite burden that correlates with the kinetics of IEC responsiveness to IFN-{gamma} stimulation. Together, our work uncovers how Cryptosporidium infection impacts the IEC compartment and helps define the relationship between the kinetics of IFN-{gamma} responsiveness and pathogen control in IEC.

immunology↗

bioRxiv2023

Intestinal cDC1s provide IL-12 dependent and independent functions required for CD4+ T cell mediated resistance to Cryptosporidium

Cryptosporidium is an enteric pathogen that is a prominent cause of diarrheal disease. Control of this infection requires CD4+ T cells, though the processes that lead to T cell-mediated resistance have been difficult to assess. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to dissect the early events that influence CD4+ T cell priming and effector function. These studies highlight that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node (mesLN) and differentiate into Th1 cells in the gut, where they mediate IFN-{gamma}-dependent control of the infection. Although type 1 conventional dendritic cells (cDC1s) were not required for initial priming of CD4+ T cells, cDC1s were required for CD4+ T cell expansion and gut homing. cDC1s were also a major source of IL-12 that was not required for priming but promoted full differentiation of CD4+ T cells and local production of IFN-{gamma}. Together, these studies reveal distinct roles for cDC1s in shaping CD4+ T cell responses to enteric infection: first to drive early expansion in the mesLN and second to drive effector responses in the gut. SummaryCryptosporidium parasites that express model antigens were generated to dissect how parasite-specific CD4+ T cells are primed and mediate effector functions required to control this enteric pathogen. cDC1s produced IL-12p40 and were required for early expansion and gut homing of CD4+ T cells. However, IL-12p40 was only required for the development of Th1 CD4+ T cell effector function in the gut.

immunology↗

bioRxiv2023

Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8+ T cell responses

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of IFN-{gamma} that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8+ T cell responses to Cryptosporidium.

immunology↗

bioRxiv2023

Genetic crosses within and between species of Cryptosporidium

Parasites and their hosts are engaged in rapid coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing C. parvum, a parasite of cattle and humans, and C. tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity. Significance statementThe parasite Cryptosporidium is a leading cause of diarrheal disease. While infection is common all around the world, young children experiencing malnutrition are impacted most profoundly, and the disease is an important contributor to early childhood mortality. This study experimentally demonstrates that different strains and even species of Cryptosporidium can recombine their genomes through sex. The progeny of such genetic crosses shows combined features of both parents, with resistance to multiple drugs being one example. Sex thus provides a critical mechanism for the parasite to rapidly adapt to changing environments and hosts. Genetic crosses as an experimental tool may also be harnessed in the future to discover the genes underlying differences in virulence, drug sensitivity, and immunogenicity between parasite isolates.

microbiology↗