Ковид: заражение легочных макрофагов
Важные данные.
Исследование патологов из Университета Сан Франциско, США, в котором показано, что уже на самых ранних этапах инфекции (через 48 часов) вирус САРС2 после попадания в легкие активно заражает там макрофаги и другие клетки миелоидного ряда. [Spoiler (click to open)]Там он обнаруживается даже раньше, чем в эпителиальных клетках легких (72 часа)
Работа сделана на тонкосрезной модели человеческих легких (донированных). Кроме этого, заражаемость подтверждена инкубированием различных популяций клеток легких (и имумнных клеток легких тоже) с вирусом, и обнаружена в образцах от больных ковидом (клетки из бронхоальвеолярной жидкости от интубированных ковидных).
Причем коронавирус миелоидные клетки не только заражает, нарушая их своевременный интерфероновый "ответ" на вторжение, но и вполне успешно в них размножается. Что подвтерждено и иммуноцитохимией, поточной цитометрией, микроскопией и ПЦР на "обратную" РНК вируса в зараженных иммунных клетках.
Параллельно они делали все то же и на вирусе гриппа. Грипп, как оказалось, в макрофагах не размножается, а только губит (как и другие клетки легких, куда может проникнуть).
Заражение иммунных клеток коронавирусом зависит от того, на сколько много на них АСЕ2 рецепторов. Что уханьский, что дельта- оба штамма оказывали похожий эффект. Далее. Зараженные макрофаги не обязательно погибают. Они могут жить и продуцировать вирус.
Авторы думают, что заражение имумнных клеток может пояснять различие в патологии ковида, в легких, и, кроме того, приводить к постковидным осложнениям, и, возможно, что вирус может в имумнных клетках долго персистировать, и они могут быть его депозитарием и даже помогать вирусу распространяться по легким и другим тканям (тк зараженные макрофаги или моноциты- они подвижные, и вирус в них может быть доставлен в другие части организма).
Так что тяжесть ковида, может обсулавливаться (после того как вирус "спустился" ниже из носоглотки), тем, что там за популяция миелоидных клеток в конкретных легких.
И это авторы акцентировались только на АСЕ2, без участия антител и, потенциально, АЗУ в заражении имумнных клеток. По их данным, выходит, что и без АЗУ возможно заражение макрофагов и моноцитов легких коронавирусом САРС2, и использвоание им их как транспортеров, что, сразу выводяит тяжесть ковида на новый уровень.
I[Spoiler (click to open)]n the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2 and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.
Using both imaging and flow cytometry, we also observed spike and dsRNA signal in lung immune cells (Fig. 1c and e), the former prevalent from the earliest 48-hour timepoint. Spike was colocalized to CD45+ ACE2+ cells and similarly dsRNA was found in these cells supporting that concept that immune cells may either be infected by SARS-CoV-2 or phagocytose the virus. Flow cytometry allowed us to further characterize spike+ and dsRNA+ immune cells (Fig. 1e). We observed significant dsRNA and spike signal in lung myeloid cells (CD45+ CD3- CD19- HLA-DR+ CD14+ cells, including interstitial macrophages, monocytes and monocyte-derived dendritic cells7) at 48-72h post-infection.
These data support that immune cells, particularly myeloid cells, have profound and early interactions with SARS-CoV-2, potentially leading to a major role in shaping the immune response. To further characterize the transcriptional influence of SARS-CoV-2 infection upon specific cell populations, during the first days of exposure, we applied single-cell RNA sequencing (scRNAseq) analysis to cells obtained at various timepoints after PCLS infection (Fig. 2). Hierarchical analysis identified clusters representing highly heterogenous lung complexity, including eight populations of non-immune cells, lymphocytes (T cells, B cells, NK), and four populations of myeloid cells. To appreciate unique changes in lung composition and gene expression induced by SARS-CoV-2, we compared lung slices infected by either SARS-CoV-2 or Influenza A virus (IAV). In response to IAV, the most profound change was a reduction in lung fibroblast and epithelial cell proportions, consistent with previous reports9. SARS-CoV-2, in contrast, produced no significant trends in lung nonimmune cell populations, compared to controls.
IAV was similarly destructive in the immune populations, producing a notable overall decrease in cells clustered in “Myeloid 1” (Extended Data Fig. 3a). Conversely, SARS-CoV-2 infection actually increased the myeloid fraction over time, increasing by almost 50% relative to controls at 72h . Aligning the scRNA-seq data on the two viral genomes revealed that the main targets for IAV infection were epithelial cells and fibroblasts , consistent with the observed loss of these populations in PCLSs.
In addition, IAV reads were also sporadically distributed in several other populations including immune cells. This may be attributable to the IAV entry receptor (sialic acid) being widely distributed on the surfaces of many cell types or perhaps to phagocytosis. SARS-CoV-2 reads by comparison were distinctly enriched in myeloid cells, even at the earliest 24h timepoint. Only a few reads were identified in non-immune cells).
To identify the subpopulation of myeloid cells targeted by SARS-CoV-2, the four myeloid clusters from Fig. 2a were combined and re-clustered, resulting in the definition of ten myeloid populations that included neutrophils, dendritic cells (DCs) and multiple subpopulations of monocytes and macrophages .
IAV reads remained sporadic across all 10 clusters. Conversely, SARS-CoV-2 tropism among these clusters was more selective with alveolar macrophages (AMs), IGSF21+ DCs, and monocytes accounting for the majority of significant viral reads. To account for the variable frequency of each population in Fig. 2e, we analyzed the SARS-CoV-2 read frequency and plotted this over time. This demonstrated that viral reads rose in unison in both myeloid and epithelial cell populations from 24 to 48h and then decreased in the myeloid populations at 72h. This could be the result of resolution of the infection in the PCLS, or an exhaustion of host cells targeted by the virus.
To investigate how SARS-CoV-2 affects human lung myeloid cells, we focused on the dominant AMs, which are also anatomically within the airspaces and so directly exposed to virus. A bronchoalveolar lavage (BAL) in the donor lungs produced a sample enriched in AMs, defined here as CD45+ CD169+ HLA-DR+10 and comprising notably few epithelial cells (<1% of live cells) (Extended Data Fig. 4b-c). AMs were analyzed for ACE2 expression , and we observed variability amongst lung donors with on average ~10-20% of ACE2+ AMs, but with one donor at ~80% (Fig. 3c). We incubated the cell population with SARS-CoV-2 and then analyzed cells by flow cytometry . After 48h with SARS-CoV-2 at MOI 0.1, we detected spike in 1- 10% of the AMs.
Somewhat surprisingly, an MOI of 1 did not significantly increase spike+ AM percentage compared to MOI 0.1, suggesting that either cells were somehow protected at higher titers-perhaps due to increase antiviral sensing and subsequent ISGs-or that a plateau was reached in the number of cells that were capable of being infected. At 48h, viability of AMs was high in both MOI groups, an effect that suggests the virus was not inducing AM cell death contrary to observations in blood monocytes from COVID-19 subjects11.
However, of the spike+ AMs, the majority were ACE2+ pointing to a specific but not obligate role for ACE2 in licensing AM viral entry. In support of this, when we used an ACE2 blocking antibody incubated with AMs 2h prior to SARS-CoV-2, we always observed a significant decrease in spike+ AMs but this was rarely complete, even despite using saturating concentrations of the ACE2 antibody.
Taken together, these data indicate that SARS-CoV-2 entry into AMs was significantly mediated by ACE2 expression and did not require epithelial cells as an intermediate host, since our BAL preparation lacked these cells. Macrophage infection by viruses such as IAV has long been described as abortive, but several studies have shown that the IAV H5N1 strain is capable of virus production in macrophages13.
To study the ability of AMs that were exposed to virus to produce and release new viruses, SARS-CoV-2 and IAV (fluorescent strain, PR8-venus) were incubated with BAL cells (MOI 0.1 or 1) as above and the resulting titer in the media was then re-assessed. As a control, the same quantities of viruses were incubated only with media. After 48h of incubation, cell-free supernatant was then collected from the control (media) and BAL groups and used to infect Vero (SARS-CoV-2) or MDCK cells (IAV). After 24h, the infection of these readout cells was assessed by spike staining and flow cytometry.
By quantifying Vero cells that are positive for spike (SARS-CoV-2) or MDCK cells positive for Venus (IAV), we could quantify the infective load of virus present in cell-free supernatant after incubation with either BAL cells or cell-free media. For SARS-CoV-2, incubating the virus with BAL cells amplified virus at both MOI 0.1 and 1 compared to incubating with media alone . We observed the opposite effect with IAV, which decreased the amount of virus present at MO1 0.1 .
SARS-CoV-2 has been continuously evolving, resulting in the emergence of several variants of concern (VOC), the most pathogenic of which has been the delta variant, which we directly compared to ancestral USA-WA1/2020 in our system. As previously done, BAL cells were infected with SARS-CoV-2 viruses for 48h. To determine SARS-CoV-2 viral production by BAL cells, we used a plaque assay on BAL supernatant.
BAL cells increased viral titer for both ancestral and delta confirming productive infection of BAL cells by these variants. We next sorted AMs from BAL (gated as CD3- CD19- EpCAM- CD45+ HLA-DR+ CD169+) before infection. After 48h infection with SARS-CoV-2 variants (Ancestral and delta) at MOI 0.1 or 1, cell-free supernatant was harvested and used for the plaque assay.
Consistent with results from BAL incubations, AM incubation with SARS-CoV-2 induced an increase of viral titer at both MOI 0.1 and 1 . Anti-viral responses (ISG induction) were increased in both variants with increasing MOI most significantly with ancestral.
Taken together, our results show that incubation of AMs with SARS-CoV-2, but not IAV, leads to a productive infection of these cells and viral propagation without cell death. To compare our findings in the isolated human lung model to clinical COVID-19 cases, endotracheal aspirates (ETA) were sampled from seven intubated COVID-19 patients and analyzed by scRNA-seq .
Clustering showed that the cellular population was predominantly composed of myeloid cells , which contained macrophages, neutrophils, and some DCs . Analysis of SARS-CoV-2 normalized expression revealed that SARS-CoV-2 reads localized mainly to macrophages, but in some cases were also found in neutrophils and T cells , similar to prior results. These patients were sampled at different times from 1 to 14 days after ICU admission, but the timing did not correlate with the quantity or SARS-CoV-2 reads..several macrophage subpopulations (phenotypes) were found to have SARS-CoV-2 reads with almost 25% of AMs being virus positive. Finally, we analyzed differential gene expression in infected versus uninfected AMs obtained from COVID-19 ETA samples.
Multiple interferon-stimulated genes (ISGs) were increased in infected compared to non-infected AMs in ETA samples , consistent with their being exposed to viruses more profoundly than neighboring cells. To ask whether ISG expression was a prominent feature of early infection, we returned to the PCLS system and compared uninfected, IAV, and SARS-CoV-2 exposure. We indeed found that AMs exposed to SARSCoV- 2 had upregulated ISGs.
However, this induction was typically ~10-fold less in SARSCoV- 2, compared to IAV infection. AMs are the sentinel immune cells in lung alveoli and relied upon to efficiently engulf and neutralize pathogens15. Here, we show that these cells are targeted by two of the most significant SARS-CoV-2 variants, leading to productive infection, viral propagation, and yet blunted interferon responses compared to IAV.
These results suggest a depot effect where protective defenses are highjacked to facilitate viral production. AMs are migratory in the alveolar spaces15 and SARS-CoV-2-infected AMs could potentially spread infection to uninvolved areas of the lung leading to catastrophic viral loading of the lung, including as discussed in the accompanying manuscript, the loading of interstitial macrophage populations. We also found multiple macrophage subpopulations with SARS-CoV-2 viral reads, and it is certainly possible that these non-AM cells are also capable of productive infections. As ACE2 is an interferon-stimulated gene16, initial infection may be self-propagating, since our results indicated a critical dependency on ACE2 expression, although other viral entry mechanisms are potentially involved.
As one recent example, in later stages of COVID-19 infection, and when antibodies have been generated, Fc gamma receptors appear to lead to monocyte infection11. In our model, the lack of antibodies likely rules out a role of Fc receptors in viral entry into AMs. It is intriguing to consider the long-lasting effects of this macrophage depot, which could be linked to non-resolving critical illness and long-term complications of COVID- 19.
In summary, by examining the very early immune events in the human lung after SARS-CoV- 2 infection, we discovered a specific tropism for lung myeloid populations and evidence of productive infection by AMs that has broad implications in unraveling the pathogenesis of severe SARS-CoV-2 infections in humans.
Исследование патологов из Университета Сан Франциско, США, в котором показано, что уже на самых ранних этапах инфекции (через 48 часов) вирус САРС2 после попадания в легкие активно заражает там макрофаги и другие клетки миелоидного ряда. [Spoiler (click to open)]Там он обнаруживается даже раньше, чем в эпителиальных клетках легких (72 часа)
Работа сделана на тонкосрезной модели человеческих легких (донированных). Кроме этого, заражаемость подтверждена инкубированием различных популяций клеток легких (и имумнных клеток легких тоже) с вирусом, и обнаружена в образцах от больных ковидом (клетки из бронхоальвеолярной жидкости от интубированных ковидных).
Причем коронавирус миелоидные клетки не только заражает, нарушая их своевременный интерфероновый "ответ" на вторжение, но и вполне успешно в них размножается. Что подвтерждено и иммуноцитохимией, поточной цитометрией, микроскопией и ПЦР на "обратную" РНК вируса в зараженных иммунных клетках.
Параллельно они делали все то же и на вирусе гриппа. Грипп, как оказалось, в макрофагах не размножается, а только губит (как и другие клетки легких, куда может проникнуть).
Заражение иммунных клеток коронавирусом зависит от того, на сколько много на них АСЕ2 рецепторов. Что уханьский, что дельта- оба штамма оказывали похожий эффект. Далее. Зараженные макрофаги не обязательно погибают. Они могут жить и продуцировать вирус.
Авторы думают, что заражение имумнных клеток может пояснять различие в патологии ковида, в легких, и, кроме того, приводить к постковидным осложнениям, и, возможно, что вирус может в имумнных клетках долго персистировать, и они могут быть его депозитарием и даже помогать вирусу распространяться по легким и другим тканям (тк зараженные макрофаги или моноциты- они подвижные, и вирус в них может быть доставлен в другие части организма).
Так что тяжесть ковида, может обсулавливаться (после того как вирус "спустился" ниже из носоглотки), тем, что там за популяция миелоидных клеток в конкретных легких.
И это авторы акцентировались только на АСЕ2, без участия антител и, потенциально, АЗУ в заражении имумнных клеток. По их данным, выходит, что и без АЗУ возможно заражение макрофагов и моноцитов легких коронавирусом САРС2, и использвоание им их как транспортеров, что, сразу выводяит тяжесть ковида на новый уровень.
I[Spoiler (click to open)]n the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2 and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.
Using both imaging and flow cytometry, we also observed spike and dsRNA signal in lung immune cells (Fig. 1c and e), the former prevalent from the earliest 48-hour timepoint. Spike was colocalized to CD45+ ACE2+ cells and similarly dsRNA was found in these cells supporting that concept that immune cells may either be infected by SARS-CoV-2 or phagocytose the virus. Flow cytometry allowed us to further characterize spike+ and dsRNA+ immune cells (Fig. 1e). We observed significant dsRNA and spike signal in lung myeloid cells (CD45+ CD3- CD19- HLA-DR+ CD14+ cells, including interstitial macrophages, monocytes and monocyte-derived dendritic cells7) at 48-72h post-infection.
These data support that immune cells, particularly myeloid cells, have profound and early interactions with SARS-CoV-2, potentially leading to a major role in shaping the immune response. To further characterize the transcriptional influence of SARS-CoV-2 infection upon specific cell populations, during the first days of exposure, we applied single-cell RNA sequencing (scRNAseq) analysis to cells obtained at various timepoints after PCLS infection (Fig. 2). Hierarchical analysis identified clusters representing highly heterogenous lung complexity, including eight populations of non-immune cells, lymphocytes (T cells, B cells, NK), and four populations of myeloid cells. To appreciate unique changes in lung composition and gene expression induced by SARS-CoV-2, we compared lung slices infected by either SARS-CoV-2 or Influenza A virus (IAV). In response to IAV, the most profound change was a reduction in lung fibroblast and epithelial cell proportions, consistent with previous reports9. SARS-CoV-2, in contrast, produced no significant trends in lung nonimmune cell populations, compared to controls.
IAV was similarly destructive in the immune populations, producing a notable overall decrease in cells clustered in “Myeloid 1” (Extended Data Fig. 3a). Conversely, SARS-CoV-2 infection actually increased the myeloid fraction over time, increasing by almost 50% relative to controls at 72h . Aligning the scRNA-seq data on the two viral genomes revealed that the main targets for IAV infection were epithelial cells and fibroblasts , consistent with the observed loss of these populations in PCLSs.
In addition, IAV reads were also sporadically distributed in several other populations including immune cells. This may be attributable to the IAV entry receptor (sialic acid) being widely distributed on the surfaces of many cell types or perhaps to phagocytosis. SARS-CoV-2 reads by comparison were distinctly enriched in myeloid cells, even at the earliest 24h timepoint. Only a few reads were identified in non-immune cells).
To identify the subpopulation of myeloid cells targeted by SARS-CoV-2, the four myeloid clusters from Fig. 2a were combined and re-clustered, resulting in the definition of ten myeloid populations that included neutrophils, dendritic cells (DCs) and multiple subpopulations of monocytes and macrophages .
IAV reads remained sporadic across all 10 clusters. Conversely, SARS-CoV-2 tropism among these clusters was more selective with alveolar macrophages (AMs), IGSF21+ DCs, and monocytes accounting for the majority of significant viral reads. To account for the variable frequency of each population in Fig. 2e, we analyzed the SARS-CoV-2 read frequency and plotted this over time. This demonstrated that viral reads rose in unison in both myeloid and epithelial cell populations from 24 to 48h and then decreased in the myeloid populations at 72h. This could be the result of resolution of the infection in the PCLS, or an exhaustion of host cells targeted by the virus.
To investigate how SARS-CoV-2 affects human lung myeloid cells, we focused on the dominant AMs, which are also anatomically within the airspaces and so directly exposed to virus. A bronchoalveolar lavage (BAL) in the donor lungs produced a sample enriched in AMs, defined here as CD45+ CD169+ HLA-DR+10 and comprising notably few epithelial cells (<1% of live cells) (Extended Data Fig. 4b-c). AMs were analyzed for ACE2 expression , and we observed variability amongst lung donors with on average ~10-20% of ACE2+ AMs, but with one donor at ~80% (Fig. 3c). We incubated the cell population with SARS-CoV-2 and then analyzed cells by flow cytometry . After 48h with SARS-CoV-2 at MOI 0.1, we detected spike in 1- 10% of the AMs.
Somewhat surprisingly, an MOI of 1 did not significantly increase spike+ AM percentage compared to MOI 0.1, suggesting that either cells were somehow protected at higher titers-perhaps due to increase antiviral sensing and subsequent ISGs-or that a plateau was reached in the number of cells that were capable of being infected. At 48h, viability of AMs was high in both MOI groups, an effect that suggests the virus was not inducing AM cell death contrary to observations in blood monocytes from COVID-19 subjects11.
However, of the spike+ AMs, the majority were ACE2+ pointing to a specific but not obligate role for ACE2 in licensing AM viral entry. In support of this, when we used an ACE2 blocking antibody incubated with AMs 2h prior to SARS-CoV-2, we always observed a significant decrease in spike+ AMs but this was rarely complete, even despite using saturating concentrations of the ACE2 antibody.
Taken together, these data indicate that SARS-CoV-2 entry into AMs was significantly mediated by ACE2 expression and did not require epithelial cells as an intermediate host, since our BAL preparation lacked these cells. Macrophage infection by viruses such as IAV has long been described as abortive, but several studies have shown that the IAV H5N1 strain is capable of virus production in macrophages13.
To study the ability of AMs that were exposed to virus to produce and release new viruses, SARS-CoV-2 and IAV (fluorescent strain, PR8-venus) were incubated with BAL cells (MOI 0.1 or 1) as above and the resulting titer in the media was then re-assessed. As a control, the same quantities of viruses were incubated only with media. After 48h of incubation, cell-free supernatant was then collected from the control (media) and BAL groups and used to infect Vero (SARS-CoV-2) or MDCK cells (IAV). After 24h, the infection of these readout cells was assessed by spike staining and flow cytometry.
By quantifying Vero cells that are positive for spike (SARS-CoV-2) or MDCK cells positive for Venus (IAV), we could quantify the infective load of virus present in cell-free supernatant after incubation with either BAL cells or cell-free media. For SARS-CoV-2, incubating the virus with BAL cells amplified virus at both MOI 0.1 and 1 compared to incubating with media alone . We observed the opposite effect with IAV, which decreased the amount of virus present at MO1 0.1 .
SARS-CoV-2 has been continuously evolving, resulting in the emergence of several variants of concern (VOC), the most pathogenic of which has been the delta variant, which we directly compared to ancestral USA-WA1/2020 in our system. As previously done, BAL cells were infected with SARS-CoV-2 viruses for 48h. To determine SARS-CoV-2 viral production by BAL cells, we used a plaque assay on BAL supernatant.
BAL cells increased viral titer for both ancestral and delta confirming productive infection of BAL cells by these variants. We next sorted AMs from BAL (gated as CD3- CD19- EpCAM- CD45+ HLA-DR+ CD169+) before infection. After 48h infection with SARS-CoV-2 variants (Ancestral and delta) at MOI 0.1 or 1, cell-free supernatant was harvested and used for the plaque assay.
Consistent with results from BAL incubations, AM incubation with SARS-CoV-2 induced an increase of viral titer at both MOI 0.1 and 1 . Anti-viral responses (ISG induction) were increased in both variants with increasing MOI most significantly with ancestral.
Taken together, our results show that incubation of AMs with SARS-CoV-2, but not IAV, leads to a productive infection of these cells and viral propagation without cell death. To compare our findings in the isolated human lung model to clinical COVID-19 cases, endotracheal aspirates (ETA) were sampled from seven intubated COVID-19 patients and analyzed by scRNA-seq .
Clustering showed that the cellular population was predominantly composed of myeloid cells , which contained macrophages, neutrophils, and some DCs . Analysis of SARS-CoV-2 normalized expression revealed that SARS-CoV-2 reads localized mainly to macrophages, but in some cases were also found in neutrophils and T cells , similar to prior results. These patients were sampled at different times from 1 to 14 days after ICU admission, but the timing did not correlate with the quantity or SARS-CoV-2 reads..several macrophage subpopulations (phenotypes) were found to have SARS-CoV-2 reads with almost 25% of AMs being virus positive. Finally, we analyzed differential gene expression in infected versus uninfected AMs obtained from COVID-19 ETA samples.
Multiple interferon-stimulated genes (ISGs) were increased in infected compared to non-infected AMs in ETA samples , consistent with their being exposed to viruses more profoundly than neighboring cells. To ask whether ISG expression was a prominent feature of early infection, we returned to the PCLS system and compared uninfected, IAV, and SARS-CoV-2 exposure. We indeed found that AMs exposed to SARSCoV- 2 had upregulated ISGs.
However, this induction was typically ~10-fold less in SARSCoV- 2, compared to IAV infection. AMs are the sentinel immune cells in lung alveoli and relied upon to efficiently engulf and neutralize pathogens15. Here, we show that these cells are targeted by two of the most significant SARS-CoV-2 variants, leading to productive infection, viral propagation, and yet blunted interferon responses compared to IAV.
These results suggest a depot effect where protective defenses are highjacked to facilitate viral production. AMs are migratory in the alveolar spaces15 and SARS-CoV-2-infected AMs could potentially spread infection to uninvolved areas of the lung leading to catastrophic viral loading of the lung, including as discussed in the accompanying manuscript, the loading of interstitial macrophage populations. We also found multiple macrophage subpopulations with SARS-CoV-2 viral reads, and it is certainly possible that these non-AM cells are also capable of productive infections. As ACE2 is an interferon-stimulated gene16, initial infection may be self-propagating, since our results indicated a critical dependency on ACE2 expression, although other viral entry mechanisms are potentially involved.
As one recent example, in later stages of COVID-19 infection, and when antibodies have been generated, Fc gamma receptors appear to lead to monocyte infection11. In our model, the lack of antibodies likely rules out a role of Fc receptors in viral entry into AMs. It is intriguing to consider the long-lasting effects of this macrophage depot, which could be linked to non-resolving critical illness and long-term complications of COVID- 19.
In summary, by examining the very early immune events in the human lung after SARS-CoV- 2 infection, we discovered a specific tropism for lung myeloid populations and evidence of productive infection by AMs that has broad implications in unraveling the pathogenesis of severe SARS-CoV-2 infections in humans.