Non Technical Summary
Cryptosporidiosis (Crypto), poses a global public health threat characterized by life-threating diarrhea in neonates, but severe disease in mature cattle and people is uncommon. Despite substantial economic losses to the cattle industry and the public, effective preventative and therapeutic interventions are lacking. This is in part due to gaps in our understanding of the factors limiting the development of clinical disease in adult cattle but increased disease susceptibility in newborn calves. The proposed study will utilize a robust culture system that mirrors in vivo infection to systematically evaluate development of protective immunity against Crypto -- permitting the development of rational and effective therapies.
Animal Health Component
Research Effort Categories
Basic
Applied
Developmental
Goals / Objectives
Cryptosporidiosis (Crypto), a disease caused by the parasite Cryptosporidium parvum (CP) in cattle, is one of the most economically significant production limiting diseases in calves and other agriculturally important food animals worldwide. In contrast to calves, Crypto in immunocompetent adult cattle results in only subclinical disease with limited, if any, long term effects. The differential immune dynamics responsible for resistance to infection in adult cattle, but susceptibility in calves, have not been fully explored. Intestinal epithelial cells (IEC), the target cell for CP, initiate innate mucosal immune responses after colonization in the small intestine.Dendritic cells (DC) then propagate the response by triggering and shaping the adaptive immune response, thereby controlling resolution of infection. In vivo, the overall nature of the adaptive response produced is directed by DC, which are influenced by interactions, or "crosstalk", with IEC.Under most conditions, neonatal DC are biased towards a TH2 immune response, instead of the strong TH1 response that is needed to clear CP. Yet, the key question remains: how do innate immune interactions (IEC-DC) in adult cattle, but not calves, induce immunity to CP? Significant insight into the pathogenesis of Crypto has been achieved using murine models; however, mice must be rendered immunodeficient for infection to be established and, even then, still do not develop clinical disease. These limitations make direct translation of this work into inherently susceptible species, namely cattle and humans, difficult. While this in part contributes to therapeutic failure, it highlights opportunities for additional research with models that utilize clinically relevant species. Thus, for effective therapies to be developed, there is a critical need to decode the impact of the immune interactions that ultimately control development of CP-specific immunity using culture systems mirroring in vivo infection in clinically relevant species. One factor that may be involved in CP immunity is interferon lambda (IFN-λ). Several of studies have demonstrated the critical role of IFN-l in mucosal immunity during infection in a number of species, and even in CP immunity in pigs, but the role of IFNλduring infection in cattle has yet to be explored. The overall objective of this work is to identify targets for CP immunotherapy, and specifically IFN-λ3, by elucidating the impact of IEC-MoDC crosstalk on the development of protective immunity to CP in adult cattle. The central hypothesis of this project is that CP-induced IEC-DC crosstalk and resultant T cell responses will induce a protective TH1 response in adult cattle with this response further augmented by exposure to IFN-λ3. In AIM 1 an ex vivo CP-exposure model will establish the impact of IEC-DC crosstalk-induced immune signatures in adult cattle following exposure to IFN-l3. Immune signatures will be determined by characterizing the following: cellular metabolism, cellular phenotype, cytokine secretion, and the capacity to activate naïve T cells. The immediate goal of this work is to elucidate the immune mechanisms underlying protective immunity to CP. In doing so, this study will identify preventative and therapeutic targets that will be evaluated for efficacy using ex vivo and in vivo models for use in calves. This study will directly contribute to the long-term goal of this project, which is to support the health and welfare of cattle. Further, the robust culture system and mechanistic approach used herein will support investigation into disease pathogenesis and therapeutic discovery for countless other diseases in cattle.
Project Methods
AIM 1: Assess the impact of IFN-λ3 on the inherent immune signature induced by IEC-DC crosstalk after ex vivo CP-exposure.AIM 1A. Assess metabolic profiles, cytokine secretion, and cellular phenotype induced by CP-exposed IEC-MoDC crosstalk in the presence or absence of IFN-λ3.Research Design. This subaim will use an IEC-MoDC co-culture system under serum-free conditions. This will allow investigation into the inherent cellular capabilities of IEC-MoDC crosstalk by eliminating the immune modulating components found in serum. AIM 1B. Evaluate IEC-MoDC induced T cell activation following CP-exposure.Research Design. MoDC co-cultured with CP- and IFN-λ3-exposed IEC will then be cultured with allogenic T cells under serum-free conditions.Justification & feasibility. Protection against CP is conferred by a predominate TH1 response. In vivo murine models have identified DC-induced T cell activation as a critical step in developing CP-specific immunity. Further, studies have demonstrated that priming of MoDC with IFN-λleads to altered cytokine-mediated T cell responses. Here, we seek to examine whether IEC-MoDC crosstalk induced by CP exposure is modulated by IFN-λ3 leading to qualitative (TH1 vs. TH2) and quantitative (proliferation, CD25 expression) differences in T cell activation. The objective of this aim is to characterize the impact of CP induced IEC-MoDC crosstalk, with and without concurrent exposure to IFN-λ3, on T cell activation in cells derived from adult cattle. To attain this objective, we will test our working hypothesis that MoDC isolated from adult cattle that are co-cultured with IEC infected with CP will demonstrate functional and phenotypic characteristics of mature dendritic cells that are able to stimulate T cells in a TH1 oriented response and that exposure of IEC to IFN-λ?3 during CP infection will enhance the immune response of MoDC. AIM 1A. IEC monolayers will be generated from enteroids generated in a 3D matrix from submucosal crypts isolated from the ileum of a single healthy mature cow using 24-well transwell inserts (0.4 μm pore size). Transepithelial electrical resistance (TEER) using a Millicell ERS2- Voltohmmeter will determine integrity and confluency of monolayer membranes. MoDC generated from magnetically purified CD2 negative monocytes in serum-free media from Holstein cows (mid-lactation, ages 5 to 10 years) will be plated as described. The apical surface of the IEC monolayer will be exposed to 1x106 excysted CP oocysts on day 3 of co-culture with MoDC with some wells also concurrently exposed to IFN-λ3. After 12 hours of exposure, cells (MoDC + IEC) and culture supernatants will be collected. Metabolic transition to aerobic glycolysis following cellular activation will be measured using an Extracellular Flux (Seahorse XFe96) Analyzer (Agilent Technologies) in a high-throughput manner as previously described. Cytokine secretion will be quantified using the Luminex MAGPIX instrument and a validated Magpix-Milliplex bovine cytokine panel for IFNγ, IL-1α, IL-1β, IL-4, IL-8, IL-10, IL-17A, MIP-1α, and TNFα (MilliporeSigma). The activation status of IEC and MoDC will be measured via flow cytometry on the Guava easyCyte 12HT (Luminex) or via immunohistochemistry using a panel of fluorescent antibodiesvalidated by our laboratory and others. A combination of PMA and ionomycin will be used as a positive control for stimulation of cells for all analyses.AIM 1B: Following a 12 h exposure time to CP, some of the MoDC described above will be harvested to serve as stimulator cells in a mixed lymphocyte reaction (MLR). Magnetically sorted CD2 positive lymphocytes isolated from a healthy, mature, un-related Jersey cow will be utilized as the responder T cells. After labeling T cells with Cell Trace Violet to assess proliferation (flow cytometry analysis of generational peaks), they will be cultured 1:1 with MoDC in a mixture of conditioned medium and medium optimized for serum-free T cell culture. T cell proliferation, cellular metabolism, cytokine secretion, cellular phenotype and cytokine secretion will be conducted as described in AIM 1A for MLR cultures; moreover, T cells will be assessed for activation by CD25 expression via flow cytometry. A combination of PMA and ionomycin will be used as a positive control for stimulation of T cells and MLR conditions for all analyses.AIM 1 Expected Outcomes: In AIM 1A, we expect IEC-MoDC co-cultures to be activated following CP exposure assessed by the above parameters. Specifically, we expect this to be demonstrated by a mitochondrial collapse and transition to aerobic glycolysis and an activated cellular phenotype (increased expression of and II, CD1w2, CD86, CD40 and CD205). Further, we expect that the cytokine profile produced by IEC-MoDC crosstalk in cattle to exhibit polarization towards a more inflammatory profile (increased IFN-γ and increased IL-10). In Aim 1B, we anticipate that IEC-MoDC cultures will effectively stimulate T cells observed by increased T cell proliferation, CD25 surface marker expression, and reprogramming of cellular metabolism. Differences in T cell cytokine production indicative of polarization towards a TH1 biased response (decreased IL-4) is expected. Collectively, these findings would suggest that IEC-MoDC crosstalk contributes to immunity to CP infection via T-cell activation. Finally, we expect that concurrent exposure of CP-exposed IEC-MoDC to IFN-λ3 will alter the immune signature of IEC and MoDC, promoting a TH1 biased response. Potential pitfalls and alternative strategies: The transmembrane pore size (0.4 m) used in this study permits transfer of soluble mediators but limits direct cell-cell contact. Although this pore size has been used in other studies demonstrating IEC-MoDC crosstalk, it is possible that no difference will be observed for IEC-MoDC co-cultures compared to IEC or MoDC cultured alone. If this were to occur, we would investigate the specific role of greater cell-cell contact by using transmembranes of different pore sizes (1 to 4 μm). Further, as sporozoites are ~4 um x 0.6 um, using 0.4 um transmembrane pore size will prevent crypto-DC direct activation. Although its role is well documented in other infectious disease models in other species, it is also possible that IFN-λ3 will not alter the immune signature of IEC, MoDC or T cells, or that exposure will produce a less desirable T cell response (TH2 or T regulatory). This finding would still be important as it would elucidate the previously undefined role of IFN-λ3 in regulating bovine immunity. Finally, while antibodies for human CCR5 have been used to target surface markers of bovine cells, using antibodies directed against human CCR6 in bovine cells has yet to be reported, so it is possible that this detection of CCR6 may not be efficient, despite comparable homology between species. Moreover, while recombinant human IFN-λ3 has been successfully used in porcine models, it is possible that this recombinant protein may not elicit a considerable response in bovine cells, despite similar homologies between cattle, humans, and pigs.