While most COVID-19 patients have mild symptoms, about 15% of patients develop severe COVID-19 often with fatal outcomes, and scientists have been on the hunt to understand the underlying immune mechanisms driving disease severity. In a recent study in Immunity, Szabo et al.¹ provide comprehensive longitudinal insights into the phenotypical and functional shifts of T cells, monocytes and macrophages driving inflammation in the lungs of patients with severe COVID-19.

COVID-19, a disease caused by the coronavirus SARS-CoV-2, has continuously been putting a toll on life worldwide since the first cases started to spread at the end of 2019. SARS-CoV-2 is highly contagious, can be transmitted via various routes including via aerosols and predominantly infects the respiratory system. Thus, the disease often manifests with symptoms similar to a classic cold or flu, such as coughing, a runny nose and fever. However, in some cases patients suffer from severe disease characterized by major dysregulation of immune responses, cytokine storm syndrome, massive lung tissue damage resulting in difficulties or inability to breathe on their own and the need for intubation and ICU care.² Over the past year, multiple studies have profiled immune cell frequencies, phenotypes and responses in the blood of patients with COVID-19 trying to correlate these to the risk of developing mild vs. severe COVID-19, in addition to other factors such as age, gender and comorbidities due to underlying medical conditions.

In recent months, it has become obvious that certain innate immune cells, namely monocytes, become dramatically dysregulated exhibiting aberrant immune functions during the course of infection with SARS-CoV-2.^{3, 4} Previous studies focused mostly on blood monocytes from COVID-19 patients with varying disease severity, thus drawing an inconclusive picture of monocyte involvement in disease pathogenesis.^2-5 This current study by Szabo et al., coming from the group of Donna Farber, however, compared paired blood and airway samples from patients with severe COVID-19, providing comprehensive insights into the potential mechanisms responsible for the massive lung damage observed in severe COVID-19. They performed longitudinal high-dimensional profiling of blood and airway monocyte and macrophage populations (as well as T cells) describing their phenotypes, transcriptomes and corresponding cytokine secretion profiles. Overall Szabo et al. found myeloid cells to be hyperinflammatory, with aberrant CD163^hiHLA-DR^loCCR2^hi monocytes increased in the blood and a reduction of CD16⁺ monocytes in the lungs of patients with severe COVID-19 (Figure 1)¹, matching previous reports by Silvin et al. and Schulte-Schrepping et al.^{3, 4}

In order to be able to correlate immune profiles during severe COVID-19 across tissues and over time, Szabo et al. collected blood samples and saline washes of the endotracheal tube which, as they had shown previously, contain most of the respiratory immune cell populations, from intubated ICU patients with severe COVID-19 for up to 10 consecutive days. By multiparameter profiling of these samples by flow cytometry, Szabo et al. detected lowered T cell frequencies but increased frequencies of aberrant monocytes in the blood of patients with severe COVID-19, in line with a few other publications.^3-5 Yet another recent report showed that the overall monocyte numbers were barely altered, or were even decreased for certain subsets, while dendritic cell (DC) numbers were generally decreased in severe patients.⁶ Disease driven changes in the immune cell compartment generally appeared more manifested in the blood than within the airways. The authors found that within the airways myeloid cell frequencies generally increased with age, while T cell frequencies were inversely correlated. Further, these frequency distributions were not only pronounced in patients with severe COVID-19 who eventually succumbed to the disease, but also were predictive of disease outcome for at least 75% of cases – an observation that could be useful for adjusting the therapy strategy for individual patients.¹

Given the diversity of myeloid cell types, from DCs to neutrophils, monocytes and macrophages to many others, the obvious question was which myeloid subsets contributed most to these changes? To address this, Szabo et al. clustered myeloid populations based on surface marker expression and identified nine main clusters that were profoundly altered within the airway and blood samples from healthy donors vs. patients with severe COVID-19. Besides a drastic reduction of CD16⁺ intermediate monocytes in the airways of COVID-19 patients, the frequencies were highly increased for CD163^loHLA-DR^lo and CD86^hiHLA-DR^hi cells in the airways and for CD163^hiHLA-DR^lo monocytes in the blood. These monocytes were not present in healthy blood controls where the airways contained mostly CD163^hi macrophages and only a few CD163⁺ intermediate monocytes, suggestive of increased monocyte infiltration from the blood stream into the airways during severe COVID-19.¹ This dramatic shift between a high expression of HLA-DR in the airways but downregulation of HLA-DR on blood monocytes is in line with other recent publications, suggesting an immature or dysregulated phenotype of monocytes and macrophages as a driver for pathogenesis during SARS-CoV-2 infection.^{3, 4, 7} In general, few publications over the past year have addressed the dysregulation of monocytes and macrophages in COVID-19. Liao et al. had found inflammatory monocyte-derived FCN1⁺ macrophages in the bronchoalveolar fluid of patients with COVID-19,⁸ while Zhou et al. observed increased frequencies of CD14⁺CD16⁺ inflammatory monocytes secreting MCP1, IP-10 and macrophage inflammatory protein-1α (MIP-1α) contributing to the cytokine storm observed in patients with severe COVID-19.⁹ These hyperinflammatory responses by myeloid cells, including a macrophage activation syndrome, during infection with SARS-CoV-2 have been attributed as a major driver of disease severity.² However, the data presented here by Szabo et al. also appear to show a dramatic shift in DC frequencies, and perhaps functions, between healthy donors and severe COVID-19 blood and airway samples, the relevance of which needs to be studied further. Looking at the overall findings of this publication and other publications showing, for example, dysregulated type I interferon responses during COVID-19^{3, 10, 11}, it would be interesting to also address the impact of DCs on COVID-19 outcome in future studies.

Another question should be how exactly do aberrant monocytes or lung macrophage populations contribute to the lung damage (or other pathologies) observed in patients with severe COVID-19? Do differently localized subsets or subsets of embryonic vs. bone marrow-derived origin contribute in different ways? In an attempt to address some of these questions, Szabo et al. performed scRNA-seq on paired blood and airway samples from patients with severe COVID-19 and showed that pro-inflammatory gene signatures were highly upregulated in airway monocytes and macrophages compared with blood monocytes, as indicated by the expression of CD206 or ITGAV. Meanwhile, blood monocytes showed increased expression of genes for “homing” receptors such as CCR2 or CX3CR1.¹ Airway myeloid cells had increased transcript levels for CCL2, which was also upregulated in airway T cells (which are mostly composed of tissue resident memory T cells becoming highly activated during severe COVID-19), which, Szabo et al. hypothesized, was facilitating enhanced recruitment of monocytes from the blood to the lungs. Similarly, Zhou et al. and others had previously shown elevated levels of CCL2 and CCL7 in the BALF from severe patients, thus increasing monocyte recruitment into the lung.^{2, 12} Matrix metalloproteinases (e.g. MMP9), which had been implicated previously in driving tissue damage, were also highly upregulated in airway myeloid cells of patients with severe COVID-19 and may contribute to pathogenesis.¹

In addition, CD163⁺ myeloid cells were aggregated in the alveolar spaces of lung autopsy samples from COVID-19 patients with diffuse alveolar damage, leading Szabo et al. to conclude that myeloid cell recruitment into the alveoli is perpetuating inflammation and pathogenesis. Although diffuse alveolar damage is one key characteristic of the pulmonary pathology exhibited by COVID-19, which massively impedes alveolar gas exchange², simply the presence of CD163⁺ myeloid cells in the alveolar spaces does not tell us much about why these cells aggregate there. Their functions could be pro-inflammatory or also aiding tissue regeneration but also anti-inflammatory, as CD163⁺ macrophages often have been described as immunosuppressive in the context of tumor microenvironment.¹³ It is intriguing to speculate that this immunosuppressive function could be the case in COVID-19, while pro-inflammatory monocytes keep “firing” cytokines resulting in a pro-anti-inflammatory loop that cannot be properly resolved in severe COVID-19. Szabo et al. identified that cytokine and chemokine gradients from the blood to the airways changed over time, with the higher levels of the monocyte chemoattractant MCP-1/CCL2, MIP-1α/CCL3 or MIP-1β/CCL4 in the airways facilitating monocyte infiltration and recruitment, which may explain the aggregation of CD163⁺ myeloid cells in alveolar spaces.¹

In conclusion, Szabo et al. provide a comprehensive data set of paired blood and airway monocytes and macrophages from patients with severe COVID-19 employing multiparameter flow cytometry and scRNA-seq, confirming their aberrant and hyperinflammatory profiles, and an increased influx of aberrant monocytes into the lung following a CCR2-CCL2 gradient, which likely give rise to alveolar damage promoting CD163⁺ myeloid cells.¹ More functional data are needed now to confirm the hypothesis built by Szabo et al., and other authors, providing more explicit mechanisms of tissue damage, specialized functions and contributions of each of the identified aberrant monocyte or macrophage subsets. This will be crucial to fully understand pathogenesis during severe COVID-19, and thus, how to prevent and/or treat it to improve disease outcome for patients, perhaps in a more personalized or cell-targeted way than the current “standard” therapies such as anti-IL-6 treatment, remdesivir or targeting of GM-CSF.^14-16

虽然大多数 COVID-19 患者症状轻微，但大约 15% 的患者会出现严重的 COVID-19，通常会导致致命的后果，科学家们一直在努力了解导致疾病严重程度的潜在免疫机制。在最近的一项免疫研究中，Szabo等人。^图1提供了关于 T 细胞、单核细胞和巨噬细胞在严重 COVID-19 患者肺部引起炎症的表型和功能变化的全面纵向见解。

COVID-19 是一种由冠状病毒 SARS-CoV-2 引起的疾病，自 2019 年底首例病例开始传播以来，它一直在给全世界的生命造成损失。SARS-CoV-2 具有高度传染性，可以传播通过各种途径，包括通过气溶胶，主要感染呼吸系统。因此，该疾病通常表现为类似于典型感冒或流感的症状，例如咳嗽、流鼻涕和发烧。然而，在某些情况下，患者患有严重疾病，其特征是免疫反应严重失调、细胞因子风暴综合征、大量肺组织损伤导致呼吸困难或无法自行呼吸，以及需要插管和 ICU 护理。²在过去一年中，多项研究分析了 COVID-19 患者血液中的免疫细胞频率、表型和反应，试图将这些与发生轻度和重度 COVID-19 的风险以及年龄等其他因素联系起来，性别和合并症由于潜在的医疗条件。

近几个月来，很明显，某些先天免疫细胞，即单核细胞，在感染 SARS-CoV-2 的过程中会显着失调，表现出异常的免疫功能。^{3, 4}以前的研究主要集中在来自不同疾病严重程度的 COVID-19 患者的血液单核细胞上，因此无法得出单核细胞参与疾病发病机制的结论。^2-5 Szabo等人的当前研究然而，来自 Donna Farber 的小组比较了来自重症 COVID-19 患者的配对血液和气道样本，提供了对导致严重 COVID-19 中观察到的大量肺损伤的潜在机制的全面见解。他们对血液和气道单核细胞和巨噬细胞群（以及 T 细胞）进行了纵向高维分析，描述了它们的表型、转录组和相应的细胞因子分泌谱。总体而言，Szabo等人。发现骨髓细胞过度炎症，血液中异常 CD163 ^hi HLA-DR ^lo CCR2 ^hi单核细胞增加，重症 COVID-19 患者肺部CD16 ^{+单核细胞减少（图 1）1}，与 Silvin等人以前的报告相匹配。和 Schulte-Schrepping等人。^3、4

为了能够在严重 COVID-19 期间跨组织和随着时间的推移关联免疫谱，Szabo等人。连续 10 天从重症 COVID-19 插管的 ICU 患者收集血样和气管内导管的盐水冲洗液，如之前所示，其中包含大部分呼吸道免疫细胞群。通过流式细胞仪对这些样品进行多参数分析，Szabo等人。与其他一些出版物一致，检测到严重 COVID-19 患者血液中 T 细胞频率降低但异常单核细胞频率增加。^3-5最近的另一份报告显示，总体单核细胞数量几乎没有改变，某些亚群甚至有所减少，而重症患者的树突状细胞 (DC) 数量普遍减少。⁶免疫细胞区室中疾病驱动的变化通常在血液中比在气道中更明显。作者发现，气道内骨髓细胞的频率通常随着年龄的增长而增加，而 T 细胞的频率呈负相关。此外，这些频率分布不仅在最终死于该病的重症 COVID-19 患者中更为明显，而且还可以预测至少 75% 病例的疾病结果——这一观察结果可能有助于调整治疗策略个别患者。¹

鉴于骨髓细胞类型的多样性，从 DC 到中性粒细胞、单核细胞和巨噬细胞再到许多其他细胞，显而易见的问题是哪些骨髓亚群对这些变化的贡献最大？为了解决这个问题，Szabo等人。基于表面标志物表达的髓样细胞群，并确定了九个主要集群，这些集群在健康供体与严重 COVID-19 患者的气道和血液样本中发生了深刻的变化。除了COVID-19 患者气道中CD16 ⁺中间单核细胞的急剧减少外，气道中 CD163 ^lo HLA-DR ^lo和 CD86 ^hi HLA-DR ^hi细胞以及 CD163 ^hi HLA-DR的频率也大大增加^lo血液中的单核细胞。这些单核细胞不存在于健康的血液对照中，其中气道主要含有 CD163 ^hi巨噬细胞，只有少数 CD163 ⁺中间单核细胞，这表明在严重的 COVID-19 期间单核细胞从血流到气道的浸润增加。¹气道中 HLA-DR 的高表达与血液单核细胞中 HLA-DR 的下调之间的这种戏剧性转变与最近的其他出版物一致，表明单核细胞和巨噬细胞的不成熟或失调表型是 SARS-期间发病机制的驱动因素。 CoV-2 感染。^{3, 4, 7}一般来说，过去一年中很少有出版物涉及 COVID-19 中单核细胞和巨噬细胞的失调。廖等人。在 COVID-19 患者的支气管肺泡液中发现了炎性单核细胞衍生的 FCN1 ^{+巨噬细胞， 8}而 Zhou等人。^{观察到分泌 MCP1、IP-10 和巨噬细胞炎症蛋白-1α (MIP-1α) 的 CD14 +} CD16 ⁺炎性单核细胞的频率增加，导致在重症 COVID-19 患者中观察到细胞因子风暴。⁹在感染 SARS-CoV-2 期间，骨髓细胞的这些过度炎症反应（包括巨噬细胞活化综合征）被认为是疾病严重程度的主要驱动因素。²然而，Szabo等人在此提供的数据. 似乎还显示出健康供体与严重的 COVID-19 血液和气道样本之间的 DC 频率和功能的显着变化，其相关性需要进一步研究。查看本出版物和其他出版物的总体发现，例如，在 COVID-19 3、10、11 期间 I 型干扰素反应失调^，在未来的研究中探讨 DC 对 COVID-19 结果的影响也会很有趣.

另一个问题应该是异常单核细胞或肺巨噬细胞群究竟是如何导致在严重 COVID-19 患者中观察到的肺损伤（或其他病理）？胚胎与骨髓来源的不同局部亚群或亚群是否以不同的方式做出贡献？为了解决其中一些问题，Szabo等人。对来自重症 COVID-19 患者的配对血液和气道样本进行 scRNA-seq，结果表明，与血液单核细胞相比，气道单核细胞和巨噬细胞中的促炎基因特征高度上调，如CD206或ITGAV的表达所示。同时，血液单核细胞显示“归巢”受体（如CCR2 ）的基因表达增加或CX3CR1。¹气道骨髓细胞的CCL2转录水平增加，这在气道 T 细胞（主要由在严重 COVID-19 期间高度激活的组织驻留记忆 T 细胞组成）中也上调，其中，Szabo等人。假设，促进单核细胞从血液到肺部的增强募集。同样，周等人。和其他人此前曾显示重症患者 BALF 中 CCL2 和 CCL7 水平升高，从而增加了单核细胞向肺的募集。^{2, 12}基质金属蛋白酶（例如MMP9)，以前与驱动组织损伤有关，在重症 COVID-19 患者的气道骨髓细胞中也高度上调，并可能导致发病机制。¹

此外，CD163 ⁺骨髓细胞聚集在来自弥漫性肺泡损伤的 COVID-19 患者的肺尸检样本的肺泡空间中，导致 Szabo等人2019年 得出结论，骨髓细胞募集到肺泡中使炎症和发病机制持续存在。尽管弥漫性肺泡损伤是 COVID-19 所表现出的肺部病理学的一个关键特征，它严重阻碍了肺泡气体交换²，但仅仅在肺泡空间中存在 CD163 ⁺骨髓细胞并不能告诉我们这些细胞为何聚集在那里。它们的功能可能是促炎或帮助组织再生，但也有抗炎作用，如 CD163 ⁺巨噬细胞通常被描述为在肿瘤微环境中具有免疫抑制作用。¹³有趣的是，推测这种免疫抑制功能可能是 COVID-19 的情况，而促炎单核细胞不断“激发”细胞因子，从而导致在严重的 COVID-19 中无法正确解决的促抗炎循环。萨博等人。发现从血液到气道的细胞因子和趋化因子梯度随时间而变化，气道中单核细胞趋化因子 MCP-1/CCL2、MIP-1α/CCL3 或 MIP-1β/CCL4 水平较高，促进了单核细胞浸润和募集，这可以解释 CD163 ⁺骨髓细胞在肺泡空间的聚集。¹

总之，Szabo等人。使用多参数流式细胞术和 scRNA-seq 提供来自重症 COVID-19 患者的配对血液和气道单核细胞和巨噬细胞的综合数据集，确认其异常和高炎症特征，以及在 CCR2- 后异常单核细胞流入肺的增加CCL2 梯度，可能导致肺泡损伤，促进 CD163 ⁺骨髓细胞。¹现在需要更多的功能数据来证实 Szabo等人建立的假设.，和其他作者，提供了更明确的组织损伤机制、特殊功能和每个已识别的异常单核细胞或巨噬细胞亚群的贡献。这对于充分了解严重 COVID-19 期间的发病机制至关重要，因此，如何预防和/或治疗它以改善患者的疾病结果，可能以比当前“标准”疗法更个性化或细胞靶向的方式，例如作为抗 IL-6 治疗、瑞德西韦或 GM-CSF 的靶向治疗。^14-16