El papel del cd163 y otros biomarcadores deinterés en la inmunopatogenia del prrsv-1 a nivel pulmonar
- Autores: José María Sánchez Carvajal
- Directores de la Tesis: Irene Magdalena Rodríguez Gómez (dir. tes.), Jaime Gómez Laguna (dir. tes.)
- Lectura: En la Universidad de Córdoba (ESP) ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Inmaculada Luque Moreno (presid.), Lluís Luján Lerma (secret.), Norbert Stockhofe Zurwieden (voc.)
- Programa de doctorado: Programa de Doctorado en Biociencias y Ciencias Agroalimentarias por la Universidad de Córdoba
- Materias:
- Enlaces
- Tesis en acceso abierto en: Helvia
- Resumen
1. Introducción o motivación de la tesis Porcine reproductive and respiratory syndrome (PRRS) is still considered one of the costliest viral diseases impacting on the modern pig industry worldwide. PRRS virus (PRRSV), the causative agent of PRRS, is also a key player in porcine respiratory disease complex (PRDC), modulating the host immune response, leading to persistent infection, and favouring secondary bacterial infections.
PRRSV replicates primarily in the lung, being the pulmonary alveolar macrophages (PAMs) the target cell for viral replication and CD163 scavenger receptor the essential mediator for viral internalisation and disassembly. PRRSV induces a mild to severe interstitial pneumonia, with modulation and impairment of pulmonary macrophage subpopulations, which may be complicated to suppurative bronchopneumonia. Since 2006, outbreaks of virulent PRRSV-1 strains have been described across Europe. They are characterised by high mortality rates, fever and severe respiratory disorders associated with an earlier and exacerbated inflammatory response with a marked tissue injury in the lung and lymphoid organs. Furthermore, PRRSV elicits a distinct immune response depending on both the virulence of the strain and the infected tissue, therefore the evaluation of target organs may pave the way to decipher the immunopathogenesis of PRRSV infection with strain of different virulence.
In the light of the above statements, the present PhD dissertation focuses on the assessment of the lesion patterns, cell death phenomena and the expression of several immune markers of potential interest, particularly the scavenger receptor CD163, in the lung of infected animals as well as the systemic immune response. Thus, this thesis aims to dissect the immunopathogenesis of the respiratory disease caused by PRRSV-1 strains of different virulence in the acute phase of infection.
2. Contenido de la investigación The first study of this PhD Thesis aims to compare the expression, distribution and kinetics of PRRSV-N-protein, CD163 and CD107a positive cells in the lung and tonsil from experimentally-infected piglets with three different PRRSV-1 strains: a virulent PRRSV-1 subtype 3 strain (SU1-bel) and two low virulent subtype 1 strains, LV and 215–06. Piglets were sequentially euthanised at 3, 7 and 35 dpi. SU1-bel strain replicated more efficiently in the lungs and tonsils. The number of CD163⁺ cells decreased in both tissues from all infected groups at 7 dpi, followed by an increase at the end of the study, highlighting a negative correlation with the frequency of PRRSV-N-protein⁺ cells. A significant increase in CD107a+ cells was observed in all infected groups at 35 dpi but no differences were observed among them. Whereas the initial decrease of CD163⁺ cells appears to be associated to virus replication and cell death, the later recovery of the CD163⁺ population may be due to either the induction of CD163 in immature cells, the recruitment of CD163⁺ cells in the area of infection, or both. These results suggest that the host is able to restore its macrophage subpopulations as well as their potential biological functions after one-month post-infection with PRRSV-1 strains, finding the greatest recovery in SU1-bel-infected animals.
In the second study, our goal was to compare the clinical signs, gross and microscopic lesions as well as the expression of CD163 within live PAM subset from BALF. Conventional piglets were intranasally inoculated with two PRRSV-1 strains of different virulence, the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6, 8 and 13 dpi. More severe clinical signs as well as higher macroscopic and microscopic lung scores were observed in Lena-infected piglets compared to 3249-infected pigs. A decreased frequency of PAMs in BALF was detected earlier in Lena group. Besides, the frequency and median fluorescence intensity of CD163 within PAMs were much lower in Lena-infected pigs than in 3249-infected pigs. This outcome may result from the effect of PRRSV replication, PRRSV-induced inflammation, the influx of immature macrophages to restore lung homeostasis and/or the evidence of CD163low cells after CD163+ cells decrease in BALF.
In the third study, we focused on evaluating Regulated Cell Death (RCD) events by means of the expression of caspases after Lena and 3249 strains infection. Lung tissue samples from the previous experiment were collected at 1, 3, 6, 8 and 13 dpi and hence, processed for their analysis. Lena virulent strain induced a significantly higher frequency of TUNEL+ cells when comparing with the frequency of cCasp3+ cells during the first week post-infection. In addition, both PRRSV-1 strains triggered cCasp8 and in a lesser extent cCasp9 after one-week post-infection together with a replenishment of CD163+ pulmonary macrophages. These results highlight the induction of several RCD pathways beyond apoptosis, such as necroptosis and pyroptosis, during the first week post-infection followed by activation of, mainly, the extrinsic apoptosis pathway during the second week post-infection. Moreover, the recovery of CD163+ macrophages at second week post-infection represents an attempt to restore pulmonary macrophage subpopulations lost during the early stages of the infection. The goal of the fourth study was to explore the role of several immune markers potentially involved during the first week post-infection in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections upon two PRRSV-1 strains of different virulence, Lena and 3249 strains. Sera and lung tissue samples were collected and accordingly processed for their corresponding analyses. Viraemia titres and clinical signs were higher in Lena-infected piglets with an increase in the sera levels of IFN-γ and IL-6 compared to 3249-infected pigs. Lena strain triggered an earlier and stronger onset of lung lesions due to extensive consolidated areas in the apical and medial lung lobes which corresponded microscopically with foci of suppurative bronchopneumonia. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals with PRRSV-N-protein+ cells associated with a marked drop in CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased upon PRRSV-1 infection, being more intense in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked in both PRRSV-1 strains at 8 dpi, highlighting a strong correlation among CD200R1+ cells and lung injury in Lena-infected pigs. These results point out the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, underlining the activation of routes potentially involved in the restraint of the local inflammatory response. Finally, the fifth study consisted in an approach to molecular histopathology by MALDI-MS Imaging with the goal of identifying new peptides involved in PRRSV-1 immunopathogenesis and lung lesion patterns. Analyses were conducted in 3 selected animals per group at 6 and 8 dpi. These time-points were selected according to the detection of marked clinical sings, gross and microscopic lung lesion scores as well as viral load in Lena- and 3249-infected piglets after previous studies. MALDI-MS Imaging study revealed a profile of 54 differentially overexpressed peptides and their putative associated proteins when comparing both infected PRRSV-1-infected groups with control group at 6 and 8 dpi. Some of these putative proteins have been previously described upon PRRSV infection, such as heat shock protein beta-1, heat shock protein 72, interferon-induced GTP-binding protein Mx1, pro-apoptotic protein BAKM variant, apolipoprotein A-IV, annexin A6, matrix metallopeptidase 9, vimentin, complement C3, filamin-A, annexin IV or cytoskeleton-associated protein 4. But also other overexpressed putative proteins, such as apolipoprotein A-I, ras-related protein Rap-1b, ribosomal protein L13, L-lactate dehydrogenase B chain, tropomyosin 1 or histone H4, were identified in our studies but not previously associated with PRRSV infection. A functionality grouped network of the overexpressed proteins created by Cluego and Cluepedia tools, showed that the majority of these putative proteins were mainly involved in “regulation of cell death” within biological processes, and in “mRNA splicing-major pathway” and “neutrophil degranulation” within reactome pathways and immune system processes, respectively.
3.Conclusión First conclusion: PRRSV-1 regulates the expression of CD163 scavenger receptor and CD107a through either a direct or indirect mechanism during the first week-postinfection, with the host being able to restore and to increase the number of cells expressing these receptors after one-month post-infection. This effect may strengthen both local immune response by the activation of lung macrophages and prime the lung against future PRRSV re-infections, as well as infections by other swine respiratory pathogens.
Second conclusion: the frequency and surface expression of the scavenger receptor CD163 decrease in live PAMs isolated from the BALF of PRRSV-1 infected piglets, being this fact earlier and stronger for the virulent Lena strain.
Third conclusion: PRRSV-1 induces the activation of non-apoptotic RCD during the first week post-infection followed by the activation of mainly extrinsic apoptosis, and in a lesser extent intrinsic apoptosis, during the second week postinfection. These events are markedly elicited by Lena strain associated with a high viral load and severe depletion of CD163+ PAMs, causing severe and early lung damage.
Fourth conclusion: Upon PRRSV-1 infection a repletion of CD163+ PAMs is elicited at two weeks post-infection, probably from CD14+ cells, which point out an attempt to reconstitute the pulmonary macrophages subpopulations lost during the early stages of the infection, contributing to resolution of pulmonary inflammation.
Fifth conclusion: Lena virulent PRRSV-1 strain induces a rise in sera concentration of IL-6 and IFN-γ associated with a higher level of viremia, pointing out to a marker of interest for monitoring virulence and lung injury (IL-6) as well as an attempt of the host innate immune response in controlling virus replication (IFN-γ).
Sixth conclusion: Virulent PRRSV-1 Lena strain-induced lung inflammatory response is regulated by the expression of CD14 and iNOS, mechanisms involved in the earlier and higher extent of lung lesion which may sensitise the lung to future secondary bacterial infections. On the other hand, the increase in the expression of CD200R1 and FoxP3 represents potential pathways activated to contain the inflammatory response.
Seventh conclusion: MALDI-MS Imaging approach enables the detection of peptide molecular signature in lung tissue upon PRRSV-1 infection with putative proteins associated with a number of pathways that might be regulated, highlighting among them the regulation of cell death, mRNA splicing-major pathway and neutrophil degranulation.
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