Periodic Reporting for period 1 - MacroRhino (Deciphering rhinovirus-mediated macrophages impairment through the establishment of human induced pluripotent stem cells-derived macrophages)
Reporting period: 2022-09-01 to 2024-08-31
A growing body of evidence suggests that viruses can modulate myeloid cell responses, leading to long-term impairment of monocyte and macrophage phenotypes. While the long-term effects of viruses directly infecting macrophages on their function and phenotype have been well-studied, the impact of viruses that primarily infect other cell types on macrophages has been largely overlooked. However, recent data indicate that virus-induced modulation of macrophages can occur even in the absence of viral replication within these cells. In fact, viruses possess broader mechanisms for influencing innate immune responses beyond those observed in permissive cells. Understanding the strategies employed by viruses to modulate macrophage responses could not only reveal potential therapeutic targets to eliminate infections but also provide avenues to prevent or mitigate virus-associated pathologies.
Human rhinovirus (HRV) has been shown to impair macrophage responses following secondary bacterial challenges. The host laboratory identified arpin as a key factor targeted by HRV, altering the phagocytic activity of macrophages, and demonstrated that HRV16-treated macrophages exhibit a "paralyzed" phenotype in terms of cytokine secretion. However, the precise mechanisms underlying the reprogramming of macrophages remain unclear. Additionally, it is not yet known whether HRV must replicate within macrophages to induce changes in their phenotype and function.
Thus, my objectives are to determine (i) whether HRV can replicate in alveolar macrophages, and (ii) to elucidate the HRV-mediated mechanisms that lead to impaired macrophage function.
Furthermore, while existing models used to study macrophages have certain advantages, they fail to fully replicate organ physiology, which plays a crucial role in shaping the phenotype of resident macrophages. To address this, I will develop a human lung alveolar macrophage-like cell (LAML) model derived from human induced pluripotent stem cells (h-iPSCs) to explore the aforementioned objectives.
Human rhinovirus (HRV) has been shown to impair macrophage responses following secondary bacterial challenges. The host laboratory identified arpin as a key factor targeted by HRV, altering the phagocytic activity of macrophages, and demonstrated that HRV16-treated macrophages exhibit a "paralyzed" phenotype in terms of cytokine secretion. However, the precise mechanisms underlying the reprogramming of macrophages remain unclear. Additionally, it is not yet known whether HRV must replicate within macrophages to induce changes in their phenotype and function.
Thus, my objectives are to determine (i) whether HRV can replicate in alveolar macrophages, and (ii) to elucidate the HRV-mediated mechanisms that lead to impaired macrophage function.
Furthermore, while existing models used to study macrophages have certain advantages, they fail to fully replicate organ physiology, which plays a crucial role in shaping the phenotype of resident macrophages. To address this, I will develop a human lung alveolar macrophage-like cell (LAML) model derived from human induced pluripotent stem cells (h-iPSCs) to explore the aforementioned objectives.
Firstly, when I arrived in the laboratory, I established a new protocol to produce HRV, as the team was encountering problems to produce the virus. Then, using hMDMs, I studied the effect of HRV on phagosome maturation and bacteria elimination. I showed that HRV16 impairs bacteria elimination in macrophages. A decrease of the hydrolytic activity and of ROS production is observed in HRV16-challenged macrophages. This phenotype is associated with a delay in phagosome maturation as shown by an increase of EEA1 and CD63 positive-phagosomes and a decrease of LAMP1 positive-phagosomes at late time points post phagocytosis. In addition, there was an increase of the total EEA1 expression in the cell and a relocalization of CD63 to cell surface. Altogether, this highlighted a perturbation of the intracellular trafficking. By RNAseq, ARL5b, a small GTPase involved in the retrograde transport from the TGN and upregulated by HRV16, was identified as a potential candidate for the observed phenotypes. By knocked-down experiments, I confirmed that ARL5b was involved in the impairment of phagosome maturation and bacteria elimination. Interestingly, in HeLa OHIO cells, a cell line permissive for HRV16 replication, ARL5b was decreased upon viral infection and acted as a restriction factor.
Following this study, I took an interest in how ARL5b expression is increased in macrophages and decreased HeLa OHIO. I showed that whereas HRV16 did not replicate in macrophages, it induced an interferon and pro-inflammatory response. Using specific neutralizing antibodies, inhibitors, and siRNA, I identified the ICAM1-PKR-ATF2 signaling axis as crucial for ARL5b induction in macrophages. Interestingly, PKR and ATF2 were not involved in the regulation of ARL5b in HeLa OHIO. Thus, PKR inhibitors represent a promising therapy for the treatment of chronic lung disease patients, and we have a filled a patent in that regard. Furthermore, I demonstrated that HRV16 triggers epigenetic reprogramming in both macrophages and HeLa OHIO cells at the ARL5b promoter. On one hand, an increase of the positive epigenetic marker H3K27Ac is observed on ARL5b promotor in macrophages. The epigenetic changes in macrophages were found to be dependent on ATF2. On the other hand, in HeLa OHIO cells, an increase of the repressive epigenetic mark H3K27Me3 was observed on ARL5b promotor. This was also associated with an increase of the expression of the methyl transferase EZH2 in HeLa OHIO.
We are currently analyzing the effect of HRV16 on gene induction upon secondary challenges (bacterial or viral). W will decipher if the impairment of the cytokines secretion previously identified in the hosting laboratory, is either due to an impairment of the gene expression, with possibly an epigenetic regulation, or to the impairment of the cytokines translation and/or secretion.
Altogether, during this project, I showed that HRV16 impairs phagosomes maturation and bacteria clearance in macrophages, in an ARL5b-dependent manner. I then showed that ARL5b induction in macrophages is dependent of the ICAM1-PKR-ATF2 signaling axis and subsequent epigenetic reprogramming. I also identified ARL5b as a restriction factor of HRV16 replication in permissive cells that is negatively regulated in an ICAM1-epigenetic reprogramming dependent manner. This experience has led so far to 2 original publications as first author (one for which I am also the corresponding author).
Following this study, I took an interest in how ARL5b expression is increased in macrophages and decreased HeLa OHIO. I showed that whereas HRV16 did not replicate in macrophages, it induced an interferon and pro-inflammatory response. Using specific neutralizing antibodies, inhibitors, and siRNA, I identified the ICAM1-PKR-ATF2 signaling axis as crucial for ARL5b induction in macrophages. Interestingly, PKR and ATF2 were not involved in the regulation of ARL5b in HeLa OHIO. Thus, PKR inhibitors represent a promising therapy for the treatment of chronic lung disease patients, and we have a filled a patent in that regard. Furthermore, I demonstrated that HRV16 triggers epigenetic reprogramming in both macrophages and HeLa OHIO cells at the ARL5b promoter. On one hand, an increase of the positive epigenetic marker H3K27Ac is observed on ARL5b promotor in macrophages. The epigenetic changes in macrophages were found to be dependent on ATF2. On the other hand, in HeLa OHIO cells, an increase of the repressive epigenetic mark H3K27Me3 was observed on ARL5b promotor. This was also associated with an increase of the expression of the methyl transferase EZH2 in HeLa OHIO.
We are currently analyzing the effect of HRV16 on gene induction upon secondary challenges (bacterial or viral). W will decipher if the impairment of the cytokines secretion previously identified in the hosting laboratory, is either due to an impairment of the gene expression, with possibly an epigenetic regulation, or to the impairment of the cytokines translation and/or secretion.
Altogether, during this project, I showed that HRV16 impairs phagosomes maturation and bacteria clearance in macrophages, in an ARL5b-dependent manner. I then showed that ARL5b induction in macrophages is dependent of the ICAM1-PKR-ATF2 signaling axis and subsequent epigenetic reprogramming. I also identified ARL5b as a restriction factor of HRV16 replication in permissive cells that is negatively regulated in an ICAM1-epigenetic reprogramming dependent manner. This experience has led so far to 2 original publications as first author (one for which I am also the corresponding author).
The results obtained from this work have led to the filing of a patent aimed at improving macrophage function in individuals with chronic pulmonary diseases. The therapeutic potential of the identified targets still needs to be validated in more complex models, including the LAML model. However, our findings hold promise for improving the quality of life of COPD patients by potentially altering treatment approaches during or after HRV infections.
Although the development of the LAML model could not be completed within the scope of this project, we are continuing its establishment in the laboratory. We plan to persist in these efforts and aim to publish the model in the coming years.
Additionally, I have overseen further ongoing studies that seek to unravel the full extent of HRV-induced modulation of macrophages. We are particularly focused on investigating the virus's role in driving epigenetic modifications within macrophages.
Although the development of the LAML model could not be completed within the scope of this project, we are continuing its establishment in the laboratory. We plan to persist in these efforts and aim to publish the model in the coming years.
Additionally, I have overseen further ongoing studies that seek to unravel the full extent of HRV-induced modulation of macrophages. We are particularly focused on investigating the virus's role in driving epigenetic modifications within macrophages.