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

Abstract

Newly emerging viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome CoVs (MERS-CoV), and H7N9, cause fatal acute lung injury (ALI) by driving hypercytokinemia and aggressive inflammation through mechanisms that remain elusive. In SARS-CoV/macaque models, we determined that anti-spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response. Alveolar macrophages underwent functional polarization in acutely infected macaques, demonstrating simultaneously both proinflammatory and wound-healing characteristics. The presence of S-IgG prior to viral clearance, however, abrogated wound-healing responses and promoted MCP1 and IL-8 production and proinflammatory monocyte/macrophage recruitment and accumulation. Critically, patients who eventually died of SARS (hereafter referred to as deceased patients) displayed similarly accumulated pulmonary proinflammatory, absence of wound-healing macrophages, and faster neutralizing antibody responses. Their sera enhanced SARS-CoV-induced MCP1 and IL-8 production by human monocyte-derived wound-healing macrophages, whereas blockade of FcγR reduced such effects. Our findings reveal a mechanism responsible for virus-mediated ALI, define a pathological consequence of viral specific antibody response, and provide a potential target for treatment of SARS-CoV or other virus-mediated lung injury.

Keywords: Cytokines; Immunoglobulins; Infectious disease; Macrophages; Pulmonology.

Conflict of interest statement

Conflict of interest: This work was financially supported by grants from the US NIH RO1HL080211 and HK RGC TRS T11-706/18-N to ZC, the TNPRC (base grant RR00164), RO1060699 to SP, HMRF 16150662, 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.

Figures

Figure 1. ADS-MVA–induced S-specific immune response enhanced pulmonary pathology in SARS-CoV–infected Chinese rhesus macaques.

(A) Experimental design used to investigate the influence of S-specific immunity on SARS-CoV–induced lung injury. Two groups of Chinese rhesus macaques (n = 8/group) were subjected to i.m. injections of ADS-MVA or control vaccine ADC-MVA at weeks 0 and 4, followed by i.n. challenge with live pathogenic SARS-CoVPUMC (1 × 105 TCID50) at 4 weeks after the second vaccination. Four animals each were sacrificed at 1 and 5 weeks after inoculation. Three healthy macaques were included as controls. (B) Serum neutralizing activity. Sera collected from macaques were tested for a 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 at the indicated time points relative to infection. (D) Pathology changes of the lung tissue. Sections were stained with H&E. D shows symptom of acute DAD exhibited in 6 of 8 ADS-MVA–vaccinated macaques with extensive exudation (yellow arrow), hyaline membranes lining the alveolar walls (black arrows), and massive cell infiltration in alveolar cavities (white arrow). Left image shows a low magnification overview (100×). Middle image shows higher magnification of the boxed area in left image (200×). Right image shows minor inflammation observed in 7 macaques received ADC-MVA (n = 8) with slight alveolar septa broadening and sparse monocyte infiltration (original magnification, 100×). (E) Histopathological scores of the ADS-MVA group, including lung samples collected at both 7 and 35 dpi, were compared against the ADC-MVA control group. See Supplemental Figure 1 for the scoring index based on severity of lung histopathology. Data represent mean ± SEM values. Statistical analysis was undertaken using 2-tailed unpaired Student’s t test. *P < 0.05, n = 4. (F) Correlation of lung histopathological scores of all macaques with sera NAb titers at 0 dpi. Solid lines denote the relationship between histopathology scores and serum neutralizing activity. Statistical analysis was performed using Spearman’s rank correlation test.

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-CoVPUMC (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.

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×).

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+CD163loCD206loHAM56–) (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 CD206hiCD68+CD163hiHAM56+ (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.

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-CoVPUMC (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).

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.

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.

Figure 8. Sera from deceased SARS patients skewed wound-healing macrophage response partially through FcγR.

In vitro–polarized alternatively activated MDM were either left unstimulated or were incubated with SARS-CoV pseudovirus alone or cocultured with SARS-CoV pseudovirus and 1–400,000 serials diluted or 1/4,000 diluted sera from deceased (n = 5) or recovered SARS patients (n = 7) for 20 hours. Cells treated with sera from patients alone served as controls. Secreted cytokine and chemokine levels were measured in the culture supernatant and are shown in data represented as a column graph (A, C, and D) and fold or percentage over supernatants from macrophages treated with virus alone (B and F). A and B show that addition of sera from deceased patient (D1) dose-dependently enhanced production of IL-8, IL-6, and MCP1 by SARS-CoV–treated alternatively activated MDM. C and D show significantly enhanced IL-8 and MCP1 production by alternatively activated macrophages treated with sera from deceased SARS patients and virus compared with cells treated with virus alone. Sera treatment alone did not induce IL-8 or MCP1 production. (E) Correlation of IL-8 production with NAb titers of sera of deceased patients. Solid lines denote the relationship between histopathology scores and serum neutralizing activity. (F) Blockade of human FcγR significantly reduced antiserum-enhanced IL-8 secretion and partially reduced MCP1 secretion. In vitro–polarized alternatively activated human monocyte–derived macrophages were cocultured with SARS-CoV pseudovirus and 1–4,000 diluted sera from deceased patient (D1) with or without FcγR blocking antibody for 20 hours. Secreted cytokine and chemokine levels were measured in the culture supernatant and are shown in data represented as increase over supernatants from macrophages treated with virus alone. Data represent mean values or mean values ± SEM of at least 3 independent experiments. Two-tailed unpaired Student’s t test and Spearman’s rank correlation test were used for statistical analysis.