Equine herpesvirus (EHV) 1 is an α-herpesvirus which is endemic in horse populations worldwide and can induce respiratory disease, reproductive disorders (e.g. abortion) and central nervous disorders (e.g. equine herpesvirus myeloencephalopathy, EHM). After inhalation of the virus, EHV1 will replicate in the upper respiratory epithelium and can subsequently spread via a cell-associated viremia towards the other target organs. Recent studies demonstrated that the majority of the EHV1-infected cells in the respiratory tissue, the draining lymph nodes and the blood were positive for the cell surface marker CD172a and have led to the assumption that EHV1 ‘hijacks’ CD172a+ cells as carrier cells to spread the virus in the host. The respiratory tract harbors several cell populations, including two highly migratory (potentially) CD172a+ cell types, namely the dendritic cell (DC) and the mesenchymal stem cell (MSC). Although MSC have been demonstrated to be CD172a+ in man and rodent, this had not yet been analyzed in horse. In addition, although DC have been shown before to be susceptible to EHV1 infection, this had not yet been investigated for MSC. Infection of DC/MSC with EHV1 may substantially affect the biological activities of these cell types. Indeed, it was previously shown that EHV1 infection of equine DC resulted in a reduced capacity of these cells to support T-proliferation. However, it was not known if, as is the case with other herpesviruses, EHV1 infection of equine DC results in a modulation of cell surface markers associated with antigen (Ag) presentation, co-stimulation and adhesion. Likewise, for equine MSC, it was unknown what the impact of a putative infection could be on the expression pattern of typical MSC cell surface markers. Therefore, the aims of this thesis were to (i) evaluate CD172a expression and EHV1 susceptibility of equine MSC, (ii) determine the effect of EHV1 infection on different cell surface markers on equine monocyte-derived dendritic cells (MDDC) and MSC, and (iii) investigate the potential involvement of viral factors and cellular mechanisms that may underlie such cell surface marker modulation.
In Chapter 1, a general introduction is provided on the classification, virion structure, and viral replication of EHV-1 and the epidemiology, prevention, and treatment of EHV1 infection. Next, we discussed the immunological response against EHV1 infection and the viral mechanisms to avoid this response. Specifically, we focused on viral factors that are of particular importance for this thesis with regard to their putative role in the modulation of cell surface proteins. We finished by discussing the potential role of the two CD172a positive cell types that were investigated in this thesis, namely the DC and the MSC, in the pathogenesis of and immune response against herpesviruses.
In Chapter 2, a general outline of the thesis is given.
In Chapter 3, we found that EHV1-infected equine MDDC showed drastic downregulation of major histocompability complex (MHC) I and CD83, substantial reduction of CD29, CD86 and CD206, and moderate lowering of CD172a. However, MHCII and lymphocyte function associated antigen (LFA) 1 were not modulated on EHV1-infected equine MDDC. In addition, virus-free supernatant of EHV1-infected MDDC was unable to induce any of the downregulations observed with wild type EHV1 and only induced a very slight upregulation of MHCI on mock-infected MDDC. Next, we evaluated the role of the viral proteins virion host-shutoff (VHS), unique long protein (pUL) 49.5, EHV1’ infected cell protein (EICP) 0 (only CD83 evaluated) and pUL56 in downregulation of cell surface proteins. We found no involvement of VHS in the downregulation of any of the evaluated cell surface markers on EHV1-infected equine MDDC, despite previous report showing that VHS is involved in downregulation of CD83 and CD86 in HSV1-infected MDDC. Also, no role for EICP0 was found in reducing CD83 on EHV1-infected equine MDDC, in contrast to the role of the HSV1 ICP0 homolog in downregulating CD83 in human MDDC. Moreover, neither pUL49.5 nor pUL56 were involved in downregulation of equine MDDC cell surface proteins, with the exception of a partial elevation of CD83 and CD86 in a pUL56 deletion mutant virus compared to wild type EHV1. For CD29 we found a minor involvement of pUL56 in EHV1-mediated downregulation.
In Chapter 4, we started to explore the cellular mechanism (mis)used by EHV1 to downregulate MHCI on equine MDDC. Despite the fact that MHCI is an EHV1 entry receptor for certain equine cell types and that other entry receptors were previously shown to be downregulated upon entry of other α-herpesviruses, we found that MHCI downregulation on equine MDDC is not a direct entry-associated event. We did, however, demonstrate that EHV1 downregulates MHCI on equine MDDC by enhancing internalization of cell surface residing MHCI, which is in line with previous findings in EHV1-infected equine fibroblasts and various Kaposi’s sarcoma herpesvirus (KSHV) infected cell types. MHCI internalization in EHV1-infected equine fibroblasts has been reported to depend on dynamin, but not clathrin and this contrasts MHCI internalization in KSHV-infected cells, which depends on both clathrin and dynamin. To evaluate the possible endocytosis mechanism(s) involved in MHCI downregulation in EHV1-infected equine MDDC, we performed a screening with several inhibitors of endocytic pathways. This led to the additional finding that EHV1 entry in equine MDDC probably depends on macropinocytosis. Furthermore, these inhibitor experiments pointed towards the involvement of clathrin-dependent endocytosis in downregulation of MHCI during EHV1 infection of equine MDDC, as we found partial reversion of MHCI downregulation upon applying inhibitors for clathrin and dynamin.
In Chapter 5, we found that equine MSC are CD172a positive, susceptible to EHV1 infection and that such infection resulted in the downregulation of select cell surface markers in a pUL56-dependent fashion. While EHV1 infection of equine MSC did drastically downregulate cell surface MHCI and substantially reduced CD29 and CD105, we found no modulation of CD44 or CD90 upon EHV1 infection. EHV1-infected equine MSC also showed a variable and moderate downregulation of CD172a. We found that virus-free supernatant of EHV1-infected MSC was unable to induce any of the modulations found after infection of MSC with EHV1 and this in contrast to human cytomegalovirus (HCMV)-mediated modulation of MSC cell surface proteins. In line with previous findings in other EHV1-infected cell types, we also found that MHCI downregulation in EHV1-infected equine MSC depends on the expression of pUL56. In addition, we found for the first time in an equine cell type that expression of EHV1 pUL56 is required for downregulation of CD29 and CD105 and contributes to downregulation of CD172a.
In Chapter 6, a general discussion is given on the results shown in this thesis.
Conclusion: In conclusion, the results from this thesis show that EHV1 infects CD172a+ cell populations, including DC and MSC, and causes downregulation of several important cell surface proteins on these infected cells. Although the exact underlying mechanisms and viral proteins involved in this downregulation need to be further defined, the results from this thesis add to our understanding of how EHV1 highjacks and manipulates its target cells for its own benefits
