Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

Anti–S-IgG induced severe lung injury during acute SARS-CoV infection.

In our previous study, i.n. inoculation of Chinese macaques with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀) led to lower respiratory tract infection in all animals within 2 days (25). However, most of them rapidly cleared infection in the lungs, and all of the animals (n = 8) exhibited mild lung lesions before 3 dpi (25). Severe lung injury in SARS-CoV–infected Chinese macaques has not been detected until 7 dpi (26, 27). To determine the effect of anti-S antibody on the extent of SARS-CoV–mediated lung injury, we adoptively transferred 5 mg (low dose) and 200 mg (high dose) purified anti–S-IgG from ADS-MVA–vaccinated but unchallenged macaques into 2 groups of unvaccinated macaques (n = 6/group) via i.v. injection, and then i.n. challenged recipients with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀) (Figure 2A). As controls, another 2 macaques were administered 200 mg of control IgG (C-IgG) derived from ADC-MVA–vaccinated macaques. We sacrificed half of the macaques in each group at 2 dpi to avoid potential disruption by virus infection–induced antibodies against nucleocapsid protein (N) and other viral proteins — and prior to when induction of ALI was typically observed. The remaining half of the macaques in each group was sacrificed at 21 dpi to evaluate long-term impact (Figure 2A). At day 2, sera from macaques that received low- or high-dose S-IgG showed neutralizing activity, with IC₅₀ values ranging from 10–260 and 1,000–10,000, corresponding to the dose of sera transfer; macaques that received C-IgG failed to show neutralizing activity (Figure 2B and Supplemental Table 1). While NAb titers in the sera of macaques in the high-dose group declined steadily between 2 and 21 dpi, macaques in the low-dose and control groups displayed increased NAb titers over time (Figure 2B), suggesting active viral replication. Indeed, SARS-CoV RNA was detected using real-time RT-PCR on oral swabs, and/or virus was recovered from lung tissue in both control animals and 5 of 6 animals (83%) in the low-dose group. Only 2 of 6 animals (33%) in the high-dose group were viral RNA⁺ (Figure 2C and Supplemental Table 1), suggesting reduced viral production by high-dose S-IgG.

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Figure 2

Anti-spike antibodies induced ALI in SARS-CoV–infected Chinese rhesus macaques.

(A) Experimental design used to investigate the influence of S-IgG on SARS-CoV–induced lung injury. Two groups of Chinese rhesus macaques (n = 6/group) were subjected to i.v. injection of high-dose (200 mg) or low-dose (5 mg) purified S-IgG from ADS-MVA–vaccinated but unchallenged macaques. As controls, another 2 macaques were administered 200 mg of C-IgG derived from ADC-MVA–vaccinated macaques. After 2 days, 3 groups of macaques were challenged i.n. with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀). Half of the animals from each group were sacrificed at 2 and 21 dpi. (B) Sera from macaques at the indicated time points were tested for the capacity to neutralize SARS-CoV pseudotype virus. (C) Detection of viral RNA in oral swabs. SARS-CoV RNA was detected by nested RT-PCR in the swabs from one of the high-dose S-IgG–treated macaques (n = 6), 3 low-dose S-IgG–treated macaques (n = 6), and 2 C-IgG–treated macaques (n = 2) at the indicated time points. Left y axis shows the viral RNA copy number per milliliter swab. Right y axis shows the serum NAb titers of each macaque at 2 dpi, which are highlighted by shaded area. (D) Pathology changes of the lung tissue (200×). Sections from macaques were stained with H&E. Images show symptom of acute DAD exhibited in macaques received high-dose and low-dose S-IgG (n = 12) with extensive exudation (red arrows), hyaline membranes (black arrows), and massive cell infiltration (yellow arrows) at 2 and 21 dpi. Right panel shows minor and moderate inflammation in the macaques received C-IgG with slight alveolar septa broadening and sparse monocyte infiltration at 2 and 21 dpi. (E) Histopathological scores of the high-dose and low-dose S-IgG groups, including lung samples collected at both 2 and 21 dpi, were compared against the C-IgG group. See Supplemental Figure 1 for the scoring index based on severity of lung histopathology.

Consistent with the findings in the control ADC-MVA–vaccinated group, histopathological examination of the lungs indicated minor and moderate inflammation at day 2 and 21 in C-IgG recipients, respectively. In contrast, all S-IgG recipients exhibited symptoms of acute DAD with various degrees of exudation, hyaline membrane formation, and hemorrhage and inflammatory cells within alveolus at both day 2 and 21 (Figure 2, D and E, and Supplemental Table 2). Of note, despite the presence of high titer sera NAbs during the chronic stage of infection (Figure 1B and Figure 2B), only 1 of 5 C-IgG and control ADC-MVA vaccine recipients showed acute DAD (Figure 1E and Figure 2E), indicating a time-restricted role for S-IgG, which mostly causes severe ALI in acutely infected macaques. Therefore, we conclude that, despite viral suppression, the presence of S-IgG at the acute stage of SARS-CoV infection caused severe ALI that persists until late stages.

S-IgG failed to prevent SARS-CoV lower respiratory tract infection and amplified IMM infiltration and accumulation in the lungs.

To determine the potential cause of S-IgG–enhanced ALI, we measured viral infection and monocytes/macrophages infiltration in the lungs and serum cytokine profile at 2 dpi. We previously found that pulmonary infection of Chinese macaques could be rapidly controlled. Viral RNA⁺ cells were detectable in the lungs of only 2 of 4 animals at 2 dpi, although all of them were viral nucleoprotein-positive (NP⁺) in the lungs and the hilar lymph nodes (LNs), where the lymphatics of the lungs drain (25). Therefore, we measured viral RNA⁺ cells by in situ hybridization (ISH), as well as NP signals in the lungs and the hilar LNs by IHC staining to evaluate the actual pulmonary infection of each macaque. Consistent with the results by viral isolation (Supplemental Table 1), viral RNA was detected in pneumocytes of 1 macaque in the high-dose (HS02, 33%), 2 macaques in the low-dose (LS01, LS02; 66%), and the C-IgG–treated macaque (Figure 3A and Supplemental Table 3), suggesting reduced productive infection by S-IgG. However, an NP antibody identified positive signals in the lungs and hilar LNs of all S-IgG and C-IgG recipients (7 of 7 macaques) but not healthy controls (Figure 3B and Supplemental Table 3), suggesting that S-IgG failed to prevent SARS-CoV entry, and SARS-CoV established lower respiratory tract infection in all of the challenged animals.

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Figure 3

SARS-CoV infection and monocytes/macrophages infiltration in the lungs of C-IgG– and S-IgG–treated macaques at 2 dpi.

(A) Representative images of SARS-CoV RNA⁺ (TRITC) and AE1/AE3⁺ (FITC) cells in the lungs of infected macaques (white arrows). The upper photo shows a low magnification overview (200×); the bottom photo shows the boxed area in the upper photo. (B) Representative images of viral protein (NP) immunostaining of the lungs and hilar lymph nodes (LN) of 7 infected and 3 uninfected animals (TRITC, white arrows) (original magnification, 200×). (C) Representative images of viral NP and monocytes/macrophages. These sections showed significantly increased IMMs in the lungs of the S-IgG group compared with the C-IgG group (MAC387⁺, CD163⁺, or CD68⁺; blue arrows). Tissue samples are double-immunostained for the SARS-CoV NP (TRITC) and markers for macrophages, including MAC387 (FITC), CD163 (FITC), and CD68 (FITC). Within the panel of representative images for the C-IgG and S-IgG groups, the left panel shows a low magnification overview; the right panel shows the boxed area in the left panel (original magnification, 200×).

Consistent with previous findings in SARS-CoV–inoculated naive Chinese macaques (n = 4) (25), double-immunostaining with the NP antibody and antibodies specific to the stages of macrophage differentiation and inflammation (MAC387, CD68, CD163) revealed significant infiltration of IMMs in the lungs of C-IgG recipients, consisting of newly infiltrated (MAC387⁺) and inflammatory (CD163⁺) monocytes/macrophages (Figure 3C and Supplemental Figure 3). The CD163⁺ cells greatly exceeded the CD68⁺ population and typically gathered around NP⁺ cells to form cell clusters (Figure 3C), showing an association between viral infection and IMM infiltration in these macaques.

Compared with C-IgG recipients, more robust signals for MAC387 and CD163 were identified in the lungs of S-IgG recipients (Figure 3C and Supplemental Figure 3), suggesting enhanced IMM infiltration by S-IgG. MAC387⁺ and CD163⁺ monocytes/macrophages not only gathered around NP⁺ cells, but also formed clusters in tissues with little NP signal (Figure 3C), suggesting that infection-induced recruitment from circulation continued after viral clearance and accumulated at the inflamed site. Moreover, more-concentrated CD68⁺ signals were found, indicating altered macrophages functional response by S-IgG treatment (Figure 3C). Consistently, elevated serum levels of IL-8 were observed in macaques treated with high-dose S-IgG at 2 dpi (Supplemental Figure 3), and the IL-8 levels are strongly correlated with anti–SARS-CoV NAb titers (Supplemental Figure 3). IL-8 is a macrophage-derived inflammatory cytokine important in initiating ALI. These results, therefore, suggest that S-IgG likely altered macrophage functional response in the lungs during acute infection, resulted in increase in IL-8 production, and enhanced ALI and IMM infiltration and accumulation.

Alveolar monocytes/macrophages assumed a wound-healing function as early as 2 dpi in macaques not treated with S-IgG.

Macrophage activation is dynamic and plastic. Different monocyte/macrophage activation statues have different specialized and critically timed roles, taking part in either the initiation and maintenance or resolution of inflammation (9). A well-organized wound-healing response leads to timely resolution of inflammation, whereas disturbance in wound-healing response results in persistent inflammation and injury. To understand the S-IgG–caused excessive inflammation, we further defined macrophage heterogeneity and activation statues in the lung lesions of C-IgG (n = 1) and S-IgG recipients (n = 3) at 2 dpi. Three healthy macaques and 4 naive Chinese macaques challenged with the same dose of SARS-CoV_PUMC and scarified at 2 dpi from a previous study were included as controls (25).

By combining 5 macrophages markers (MAC387, CD68, CD163, HAM56, and CD206) (28–30), 2 subpopulations of resident macrophages were shown in the lungs of Chinese rhesus macaques at steady state as previously described (29). They are interstitial macrophages (IMs; MAC387⁺HAM56^–CD206^–) (Figure 4A) and resident alveolar macrophages (AMs; CD68⁺ HAM56^–) expressing low levels of CD163 and CD206, a marker specific to the macrophage wound-healing response, corresponding to their intrinsic antiinflammatory function (Figure 4B) (29). In lung lesions of both C-IgG–treated and naive macaque groups challenged with SARS-CoV_PUMC, IMMs were further characterized and divided into 2 subpopulations. One subpopulation was the recently recruited MAC387⁺HAM56^–CD68^– monocytes (Figure 4C) (29), which is like IM but is larger in size and primarily coexpressed CD163 (Supplemental Figure 4). The other subpopulation was single CD163⁺ inflammatory macrophages that had differentiated from newly recruited MAC387⁺ monocytes/macrophages (Figure 4D and Supplemental Figure 4) (29). Importantly, resident AMs (CD68⁺) differentiated into HAM56⁺ cells, with a slight increase in numbers and increased size, with stronger CD163 and — more importantly — CD206 staining, suggesting that they are alternatively activated (Figure 4D). Strikingly, elevated levels of TGF-β expression were detected in all MAC387⁺ — and most enlarged CD163⁺ — as well as CD68⁺ macrophages in the lungs of both C-IgG–treated and unvaccinated macaques compared with healthy controls (Figure 4, G–I), indicating that many IMMs and activated resident AMs have assumed a wound-healing function in infected lungs within 2 days of infection.

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Figure 4

Comparison of monocyte/macrophage phenotype and function in the lungs of S-IgG– and C-IgG–treated macaques.

(A–F) Phenotype of monocytes/macrophages subpopulations in the lungs of healthy, C-IgG–, and S-IgG–treated macaques (original magnification, 200×). These sections were triple-immunostained with antibodies for CD206 (cyan), HAM56 (FITC), and MAC387 (TRITC) (A, C, and E); CD206 (cyan), HAM56 (FITC) (B, D, and F), and CD68 (TRITC) (upper panel in B, D, and F); or CD163 (TRITC) (lower panel in B, D, and F). A and B show 2 subpopulations at steady states, including interstitial macrophages (IM) (MAC387⁺HAM56^–CD206^–) (yellow arrow), and resident alveolar macrophages (AM) (CD68⁺CD163^loCD206^loHAM56^–) (white arrows). C and D show the presence of alternatively activated resident AM and accumulated IMMs in the C-IgG group. Alternatively activated AMs are CD206^hiCD68⁺CD163^hiHAM56⁺ (D, white arrows). IM are MAC387⁺HAM56^–CD206^– (C, yellow arrow). IMMs include newly infiltrating monocytes/macrophages (MAC387⁺) (C, green arrow); and inflammatory macrophages (CD163⁺CD68^–CD206^–HAM56^–) (D, green arrow). E and F show significantly increased number of IMMs and decreased number of alternatively activated macrophages in the S-IgG group. IMMs include newly infiltrating monocytes/macrophages (MAC387⁺) (E, green arrow) and inflammatory macrophages that are CD68⁺HAM56^– (green arrow, F, upper panel) and CD163⁺ CD206^–HAM56^– (green arrow, F, lower panel). Resident AMs are no longer expressing CD206 (CD68⁺HAM56⁺CD163⁺CD206^–) (blue arrow, F, upper panel). (G–J) TGF-β and IL-6 expression in macrophages in the lungs. These sections were double-immunostained with antibodies for TGF-β or IL-6 (TRITC) and MAC387, CD163, or CD68 (FITC). G shows low expression levels of TGF-β and IL-6 in IMs (MAC387⁺) and AMs (CD68⁺) in healthy macaques. H shows increased TGF-β expression in the C-IgG group (white arrows) but increased IL-6 in the S-IgG group (blue arrows). I and J show the numbers of TGF-β⁺ and IL-6⁺ monocytes/macrophages in 3 groups in a 200× field.

S-IgG treatment skewed wound-healing macrophage response in the lungs during acute SARS-CoV infection.

Compared with the macaques not treated with S-IgG, the number of IMMs (MAC387⁺ and CD163⁺HAM56^–) significantly increased in S-IgG recipients (Figure 4, E and F). Moreover, many recently recruited MAC387⁺HAM56^– monocytes/macrophages did not coexpress CD163 (Supplemental Figure 4), representing the newest recruited blood monocytes, as a result of enhanced monocytes infiltration by S-IgG. Importantly, the majority of CD163⁺HAM56^– IMMs coexpressed CD68 (Supplemental Figure 4), suggesting a change of function of this subpopulation by S-IgG. Moreover, resident macrophages (CD68⁺HAM56⁺) in the lungs of S-IgG recipients no longer express CD206, which — being surrounded by many CD163⁺ IMMs (Figure 4F) — suggests a loss of wound-healing function and a role in recruiting blood monocytes. Consistently, S-IgG–treated macaques displayed a loss of TGF-β and high expression of IL-6 in all macrophage subpopulations (Figure 4, G, H, and J). IL-6 favors macrophage accumulation at the site of injury and is important for the development of persistent inflammation and ALI (31, 32). Therefore, we conclude that, while AMs underwent phenotypic and functional changes in acutely infected lungs, many monocytes/macrophages assumed a wound-healing function for inflammation resolution, but the presence of S-IgG skewed the wound-healing response, leading to uncontrolled inflammation and tissue injury.

Onset of an antibody response prior to viral clearance is associated with abrogated wound healing responses and increased IMM lung infiltration in unvaccinated Chinese rhesus macaques.

To understand the mechanism underlying the time-restricted role of S-IgG in promoting ALI, we conducted a temporal analysis of the productive viral infection, antibody response, and macrophage functional changes in unvaccinated Chinese macaques during the first week of infection. Naive macaques were challenged i.n. with 1 × 10⁵ TCID₅₀ SARS-CoV_PUMC and sacrificed at 2, 3, and 7 dpi (4 macaques/group) (Figure 5A) (25). Successful lower respiratory tract infection was confirmed by detection of viral NP using IHC staining in the lungs of all macaques (25). Viral RNA⁺ alveolar pneumocytes were primarily found at 2 dpi (2 of 4 macaques), and infrequently at 3 dpi (0 of 4 macaques; Figure 5B and Supplemental Table 4) (25). At 7 dpi, viral RNA was detectable in the lungs of 2 macaques, AD0516 and AD0518, suggesting productive infection there (Figure 5, B and C). Only few scattered NP⁺ cells were found in AD0517 (Figure 5D). Neither NP nor viral RNA was found in the lungs or swabs of AD0515, suggesting clearance of productive viral pulmonary infection in this macaque (Figure 5D and Supplemental Table 4). Serum NAb was not observed at 2 and 3 dpi until 7 dpi in 2 of 4 macaques, AD0515 and AD0516 (Supplemental Table 4 and Figure 5E).

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Figure 5

Dynamics of the viral replication, antibody response, and macrophage activation during acute SARS-CoV infection.

(A) Experimental scheme. A total of 12 Chinese rhesus macaques were challenged i.n. with SARS-CoV_PUMC (1 × 10⁵ TCID₅₀). Four animals each were sacrificed at 2, 3, and 7 dpi. (B) SARS-CoV RNA detection by ISH in the lungs at 2, 3, and 7 dpi (original magnification, 200 ×). Viral RNA⁺ (TRITC) type I pneumocytes (FITC) was found at 2 dpi (n = 2) and 7 dpi (n = 1). (C) Viral RNA in the swabs and lung homogenates on 7 dpi. SARS-CoV RNA was detected using nested RT-PCR in swabs from 3 macaques (AD0516, AD0517, and AD0518) and lung homogenates from macaque AD0516. (D) Viral antigen and inflammatory infiltrates in the lungs at 7 dpi (200×). These sections were stained for SARS-CoV NP (red) and nucleus (blue) by IHC, showing NP signal in macrophage-like cells in AD0516 and AD0517 (red arrows), and type I pneumocytes in AD0518 (blue arrows), but substantial inflammatory infiltrates were only observed in AD0516 (green arrows). (E) Serum neutralizing activity. Serum neutralizing activity was detected in macaques AD0515 and AD0516 at 7 dpi. (F–I) Decreased wound-healing response in macaques with productive pulmonary viral infection and serum neutralizing activity (200×). These sections were double-immunostained for TGF-β (TRITC) and CD163 (FITC) or triple stained for CD206 (cyan), HAM56 (FITC), and CD163 (TRITC). The figure shows that wound-healing response took place within 2 dpi, with alternatively activated monocytes/macrophages (TGF-β⁺ and CD206⁺, white arrows) and IMMs (CD163⁺CD206^–, green arrows) coexisting in the lungs (F). After viral clearance, IMMs diminish at 3 dpi (G), and the homeostasis was restored at 7 dpi (AD0515) (H). When pulmonary viral infection persists, IMM (CD163⁺CD206^–, green arrows) infiltration and accumulation is enhanced, and wound-healing macrophages (CD206⁺) are reduced in macaques that have faster NAb response (AD0516) (I).

In agreement with the findings in the C-IgG group, temporal analysis of macrophage heterogeneity revealed significantly increased number of IMMs (CD163⁺CD206^–) in viral RNA⁺ lungs at 2 dpi (Figure 5F, right image). The IMMs diminished at 3 dpi (4 of 4 macaques) (Figure 5G) and became nearly completely absent at 7 dpi in macaques without productive pulmonary viral infection (2 of 4 macaques, AD0515 and AD0517) (Figure 5H), indicating that inflammatory response resolved with 7 days following the control of productive infection. Critically, wound-healing macrophages (CD163⁺CD206⁺ and CD163⁺TGF-β⁺) were observed in the lungs of all macaques at 2 dpi, despite productive pulmonary viral infection in macaques AD0506 and AD0508 (Figure 5F). In contrast, macaque AD0516, with serum NAbs and productive virus infection in the lungs, displayed a significantly enhanced proinflammatory macrophage response and reduced inflammatory-resolving macrophages (Figure 5I), which was not observed in macaques with productive viral infection but undetectable NAb responses (AD0518) or in macaques with detectable NAbs but undetectable viral replication (AD0515) (Figure 5D and Supplemental Table 4). Therefore, persistent viral replication and concomitant NAb detection is associated with increased IMM lung infiltration and reduced wound-healing macrophage numbers.

S-IgG modifies functional response of alternatively but not classically activated macrophages to SARS-CoV infection.

To determine whether S-IgG skews wound-healing response through modifying wound-healing macrophage functional response during SARS-CoV infection, we treated in vitro–polarized alternatively activated human monocyte–derived macrophages (MDM) for 20 hours with SARS-CoV pseudovirus and heat-inactivated sera of high-dose S-IgG–treated macaques (S-IgG sera) collected at 2 dpi. We then measured the concentrations of 13 inflammatory cytokines in cell culture supernatants, including IL-1β, IFN-α, IFN-γ, TNF-α, MCP1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33. Sera from C-IgG recipients or healthy macaques were included as controls. In vitro–polarized classically activated MDM was also studied and compared with alternatively activated MDM in parallel. Consistent with previous report, unstimulated classically activated MDM produced high levels of IL-8 and IL-6; alternatively activated MDM only produced minimal levels of those cytokines (Figure 6A). Addition of SARS-CoV pseudovirus induced IL-8 production in alternatively activated MDM to levels comparable with those found in unstimulated, classically activated MDM, as well as in low-level MCP1 and IL-6 production (Figure 6B). By contrast, SARS-CoV pseudovirus caused less than a 1-fold increase in IL-8 secretion, but not other cytokine secretion, by classically activated MDM (Figure 6C). Strikingly, while none of the sera samples induced cytokine production when they were administered into MDMs alone, administration of S-IgG sera caused a dose-dependent increase in the production of all 3 inflammatory cytokines in SARS-CoV–stimulated alternatively activated MDM, resulted in a greater than 10-fold increase in IL-6, an 8-fold increase in MCP1, and a 5-fold increase in IL-8 (Figure 6, D and E). This enhancement, however, was not observed when S-IgG sera were replaced with sera from C-IgG recipients or healthy macaques (Figure 6, F, G, and H), suggesting that S-IgG, but not other sera components, enhanced SARS-CoV–stimulated cytokine production by alternatively activated MDM. Administration of S-IgG sera had no effect on cytokine production by SARS-CoV–treated classically activated MDM (Figure 6, I, J, and K), indicating that S-IgG do not modulate virus-mediated classically activated macrophage functional response. MCP1 and IL-8 are key cytokines that regulate monocytes/macrophages migration and promote neutrophils infiltration, while IL-6 promotes persistent inflammation and injury. These results suggest that, in response to SARS-CoV infection, wound-healing macrophages produce IL-8 and low amounts of MCP1 to recruit neutrophils and blood monocytes, whereas the presence of S-IgG can result in 5- to 10-fold increases in IL-8, MCP1, and IL-6 production by wound-healing macrophages, leading to significantly enhanced and persistent monocytes infiltration and tissue injury. Critically, in the presence of Fcγ receptor (FcγR) blocking antibody, IL-8 was significantly reduced when cells were treated with SARS-CoV pseudovirus and sera from high-dose S-IgG–treated macaques (diluted 1:4,000), indicating that anti–SARS-CoV antibody modifies wound-healing response partially through FcγRs (Figure 6L). Therefore, we conclude that S-IgG directly modifies functional response of alternatively activated macrophages during acute infection, thus skewing wound-healing response, leading to hypercytokinemia, aggressive inflammation and severe lung injury.

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Figure 6

S-IgG significantly amplified proinflammatory cytokine production by SARS-CoV–treated alternatively activated macrophages.

In vitro–polarized alternatively activated macrophages were either left unstimulated or were incubated with SARS-CoV pseudovirus alone or cocultured with SARS-CoV pseudovirus and diluted sera from the high-dose S-IgG group (n = 6) or C-IgG group (n = 2) collected at 2 dpi or from healthy macaques (n = 2) for 20 hours. Secreted cytokine and chemokine levels were measured in the culture supernatant and are shown in data represented as a column graph (A–D and F–K) and fold or percentage over supernatants from macrophages treated with virus alone (E and L). A shows the levels of IL-8 and IL-6 in the supernatant of classically activated and alternatively activated MDM before treatment. B and C show that SARS-CoV treatment induced MCP1, IL-8, and very low levels of IL-6 production by alternatively activated MDM, as well as enhanced IL-8 production by classically activated MDM. D and E show that sera from S-IgG recipients caused dose-dependent increase in cytokine production from SARS-CoV–treated alternatively activated MDM. F–H show that virus treated alternatively activated MDM (VT), but not sera treated or untreated cells (UT), secreted proinflammatory cytokines IL-8 (F), MCP1 (G), and very low levels of IL-6 (H). Addition of sera (1:4,000 dilutions) from high-dose S-IgG (HST), but not C-IgG treated macaques (CT) or healthy controls (HCT), significantly amplified IL-8 (F), MCP1 (G), and IL-6 (H) production. I–K show that addition of sera (1:4,000 diluted) from high-dose S-IgG–treated (HST), C-IgG treated macaques (CT), or healthy controls (HCT) had no effect on IL-8 (I), MCP1 (J), and IL-6 (K) production by SARS-CoV–treated classically activated MDM. L shows that blockade of FcγR significantly reduced antiserum-enhanced IL-8 secretion and partially reduced MCP1 secretion. Data represent mean values or mean values ± SEM of at least 3 independent experiments. Two-tailed unpaired Student’s t test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Deceased SARS patients displayed higher levels of serum NAbs, accumulated proinflammatory, macrophages infiltration, and absence of wound-healing macrophages in the lungs.

To examine the role of S-IgG in promoting ALI in a clinical setting, we investigated antibody responses during the early stage of infection in 2 groups of deceased patients or individuals recovered from SARS and characterized the heterogeneity of monocytes/macrophages in the lungs of 3 deceased SARS patients. H&E staining of lung specimens derived from 3 deceased SARS patients revealed severe ALI (Figure 7A). Consistent with the macaque findings, IHC analysis revealed significant infiltration of CD163⁺ monocytes/macrophages into the lungs of patients, with little TGF-β expression and loss of the wound-healing marker CD206⁺, indicating predominantly proinflammatory macrophage activation and reduced inflammatory-resolving response (Figure 7B). Moreover, significantly higher levels of anti-S NAbs were detected in the sera of deceased patients (n = 6) during acute infection compared with recovered patients (n = 8) (Figure 7C). These results, therefore, are consistent with findings in experimental macaques.

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Figure 7

NAb response and phenotype analysis of accumulating monocytes/macrophages in the lungs in deceased SARS patients.

(A) Pathology changes of the lung tissue. Sections from deceased SARS patients were stained with H&E. A is representative of 3 patients (original magnification, 100×), showing symptom of acute DAD with massive cell infiltration in alveolar cavities (blue arrow). (B)Massive accumulation of IMMs and absence of wound-healing macrophage response in the lungs of deceased patients. These sections were triple-immunostained with antibodies for CD206 (cyan), HAM56 (FITC), and CD163 (TRITC) or double-immunostained with TGF-β (TRITC) and CD163 (FITC) (original magnification, 200×). The right panel in the left image shows single colors from the left image (200×). These representative images show accumulation of IMMs (CD163⁺CD68^–CD206^–HAM56^–, white arrows) and the absence of wound-healing response, indicated by loss of the signal for CD206 (cyan, B1) and TGF-β (TRITC, B2) in the lungs. (C) Serum-neutralizing activity against SARS-CoV. Sera collected from deceased (red, n = 6) or recovered (blue, n = 8) SARS patients at the early stage of infection were tested for the capacity to neutralize SARS-CoV pseudotype virus. Two-tailed unpaired Student’s t test was used for statistical analysis.

Animals, tissues, and SARS-CoV infection.

SARS-CoV–infected animals and control animals were all adult female Chinese rhesus macaques. A total of 42 Chinese rhesus macaques were challenged i.n. with a live pathogenic SARS-CoV (PUM, TCID₅₀ = 50) as previously described (25). An additional 3 uninfected macaques were inoculated with PBS as negative controls. Animals were subjected to daily measurement of anal temperature, routine blood assays, and chest radiography. Animals were euthanized at 2, 3, 7, 21, and 35 days after inoculation (Figure 1, Figure 2, and Figure 6). All animals were humanely euthanized with an i.v. overdose of pentobarbital and were immediately necropsied. Lung tissues (left and right cranial, middle and caudal lobes, and right accessory lobe) were collected and fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5 μm.

Passive transfer of IgG.

SARS-CoV S protein–immune serum was generated by vaccination with ADS-MVA vectors. IgG was purified from pooled sera by protein G affinity chromatography to >95% purity. IgG was depyrogenated, concentrated to 20–30 mg protein ml⁻¹, and administered i.v. Recipients received 200 mg or 5 mg of purified IgG per kg body weight at 48 hours before the viral challenge.

Neutralization.

A neutralization assay was established to determine the humoral immune responses generated by the vaccine as previously described (51). The neutralizing activity of heat-inactivated animal sera was determined using a pseudotype viral entry assay. The pseudotype virus was generated through cotransfection of 293T cells with 2 plasmids, pcDNA-Sopt9 and pNL4-3Luc_Env_Vpr_, carrying the optimized S gene and a human immunodeficiency virus type 1 backbone, respectively. The serial diluted serum samples were incubated with equal amounts of pseudotype virus at 37°C for 1 hour. The serum-virus mixtures were subsequently added into preseeded HEK 293T-ACE2 cells. After 56 hours, infected cells were lysed to measure luciferase activity.

Nested RT-PCR and isolation of SARS-CoV.

Pharyngeal swab samples were collected from infected monkeys on the first day after inoculation and subsequently tested for SARS-CoV using nested RT-PCR as previously described (25). Briefly, RNA was isolated using TRIZOL (Invitrogen). RT-PCR was performed in a 50 μl reaction volume with outer primer pair (5′-GCTGCATTGGTTTGTTATATCGTTA-TGC-3′) and (5′-ATACAGAATACAT-AGATTGCTGTTATCC-3′), inner primer pair (5′-TCACTTGCTTCCGTTGAGGTAGC-CAGCGTGGT-GGTTCATACAA-3′) and (5′-GGTTTCGGATGTTACAGCGTCTCCCGG-CAGAAAGCTGTAAGCT-3′). All PCR products were verified by nucleotide sequencing. SARS-CoV was isolated as previously described. Briefly, pharyngeal swab samples from the infected macaques were inoculated onto Vero-E6 cells and cultured in DMEM (Thermo Fisher Scientific). Both cytopathic effect (CPE) and immunofluorescence assay (IFA) (incubation with 1:10 dilution of SARS patient serum) were used to determine the infection status of the animals.

Double and triple immunofluorescence staining on paraffin sections.

The samples were deparaffinized and rehydrated. After blocking with normal goat serum for 30 minutes at room temperature, a rabbit anti–SARS-CoV nucleocapsid antibody (ss-006-0100, eENZYME) was applied at 4°C overnight, followed by an Alexa Fluor 448 conjugated goat anti–rabbit IgG antibody for 1 hour at room temperature. Additional immunofluorescence staining was subsequently performed by overnight incubation with primary antibodies, followed by the detection of signals using appropriate fluorescent secondary antibodies (MilliporeSigma) for 1 hour at room temperature.

Primary antibodies for immunofluresence staining.

The following primary antibodies were used in this study: SARS-CoV NP (ss-006-0100, eENZYME); MAC387 (clone MAC387, AbD, Serotec); CD68 (clone KPI, DAKO; ab125047, Abcam); CD163 (clone EDHu1, AbD and ab183476, Abcam); HAM56 (clone HAM56, DAKO); CD206 (HPA004114, MilliporeSigma); TGF-β (sc-146, Santa Cruz Biotechnology Inc.); IL-6 (sc-1265, Santa Cruz Biotechnology Inc.); AE1/AE3 (clone AE1/AE3, DAKO).

ISH studies.

ISH was developed for the detection of SARS-CoV. DNA fragments of N and S proteins from the SARS-CoV strain from Hong Kong ({"type":"entrez-nucleotide","attrs":{"text":"AY278491","term_id":"30023963","term_text":"AY278491"}}AY278491) were used as templates to generate RNA probes. The probes were labeled with digoxigenin (GENEWIZ Inc.). After deparaffinization and rehydration, the sections were treated with microwave heating in an 800 W domestic microwave oven (NN-ST556M, Panasonic) at medium power for 10 minutes in 0.01 M sodium citrate buffer. Hybridization was performed at high stringency with 150 ng/ml of denatured probe at 45°C for 16 hours in 50 μl of hybridization mix (50% formamide, 10% dextran sulfate in 2× saline sodium citrate [SSC]) in a moist chamber. Excess probe was removed by washing twice in 2× SSC for 20 minutes at 45°C, followed by 2 washes in 1× SSC and 0.1× SSC for 20 minutes at 45°C. Immunological detection was conducted using the DIG Nuclear Acid Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions. Briefly, after incubation with 1% blocking reagent for 30 minutes, the sections were incubated with sheep anti-digoxigenin alkaline phosphatase conjugate (11093274910, Roche Diagnostics) diluted at 1: 500 for 2 hours at room temperature. After 3 separate washes with washing buffer (0.1 M maleic acid, 0.15 M NaCl [pH 7.5], 0.3% Tween 20), the signal was developed using 200 μl of a HNPP/Fast Red TR mix (1758888, Roche Diagnostics) in the dark for 45 minutes. The sections were counterstained with DAPI. Tissue sections incubated with sense RNA probe were used as a negative control.

Confocal microscopy.

Confocal microscopy was performed using a Carl Zeiss LSM 700 confocal microscope equipped with 4 lasers (Zeiss). ZEN (Zeiss) was used to assign colors to the 4 channels collected: Alexa Fluor 568 and HNPP/Fast Red, which fluoresces when exposed to a 568-nm wavelength laser and appears red; Alexa Fluor 488 (Molecular Probes), which appears green; Alexa Fluor 647 (Molecular Probes), which appears cyan; and the differential interference contrast (DIC) image, which is in gray scale. The 4 channels were simultaneously collected. To differentiate between individual cells, Hoechst 33258 (nuclear marker; Molecular Probes) was used at 1 μg/ml and incubated for 5 minutes, followed by rinsing with water. The colocalization of antigens was demonstrated by the addition of colors as indicated in the figure legend.

Monocyte isolation, differentiation, and polarization of macrophages.

Monocytes were purified from peripheral blood mononuclear cells obtained from blood buffy coats (provided by Red Cross, Hong Kong, China) or monkey peripheral blood, as we previously described (52). Monkey peripheral blood was obtained in heparinized vacutainer collection tubes from healthy female rhesus monkeys. To generate M1 macrophages, monocytes were differentiated in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with GM-CSF (400 IU/ml), 2 mM glutamine, 10% decomplemented FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) for 4 days, followed by exposure to fresh medium supplemented with 5% FCS and containing GM-CSF and LPS (100 ng/ml) + IFN-γ (20 ng/ml) for an additional 72 hours. To generate M2 macrophages, monocytes were differentiated in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with M-CSF (50 ng/ml), 2 mM glutamine, 10% decomplemented FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin for 4 days, followed by exposure to fresh medium supplemented with 5% FCS and containing M-CSF (50 ng/ml) + IL-4 (20 ng/ml) for an additional 72 hours.

Quantification of cytokine levels.

A panel of 13 cytokines, including IL-1β, IFN-α, IFN-γ, TNF-α, MCP1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 in monkey sera and in culture medium were quantified using multiplexing laser bead technology (BioLegend). To assess the cytokine production of macrophages, in vitro–polarized cells were washed 3 times with PBS and were subsequently activated with SARS-CoV pseudotype virus in RPMI 1640 medium (Thermo Fisher Scientific) with or without administration of highly diluted and heat-inactivated (56°C) sera from macaques or SARS patients. FcγR-specific mouse monoclonal antibody (5 μg, clone 3G8, anti-hCD16, 16016682; clone FLI8.26, anti-hCD32, 16032981; and clone 10.1, anti-hCD64, 16064981 [BD Pharmingen]) was used for blockade of human FcγR. The supernatant of these cultures was recovered after 20 hours of stimulation, and the cytokine levels were measured and analyzed.

Statistics.

All statistical analyses were performed using the 2-tailed Student’s t test. P values less than 0.05 were considered statistically significant. Data are presented as the mean values ± SEM of at least 3 independent experiments unless otherwise indicated.

This work was financially supported by grants from the U.S. NIH RO1HL080211 and Hong Kong Research Grants Council TRS T11-706/18-N to ZC, the TNPRC (base grant RR00164), RO1060699 to SP, HMRF16150662, and the University Development Fund/Li Ka Shing Faculty of Medicine Matching Fund of the University of Hong Kong to its AIDS Institute and Hong Kong RGV. The authors would like to thank D.D. Ho and K. Liu for support and scientific advice, and Wenjie Yu for assistance of S-IgG and C-IgG purification.