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. 2021 Apr 28:12:661052.
doi: 10.3389/fimmu.2021.661052. eCollection 2021.

SARS-CoV-2 Induces Lymphocytopenia by Promoting Inflammation and Decimates Secondary Lymphoid Organs

Qun Xiang  1 , Zeqing Feng  1 , Bo Diao  2 , Chao Tu  3 , Qinghua Qiao  4 , Han Yang  1 , Yi Zhang  1 , Gang Wang  2 , Huiming Wang  5 , Chenhui Wang  1 , Liang Liu  6 , Changsong Wang  7 , Longding Liu  8 , Rong Chen  3 , Yuzhang Wu  1 , Yongwen Chen  1
Affiliations

SARS-CoV-2 Induces Lymphocytopenia by Promoting Inflammation and Decimates Secondary Lymphoid Organs

Qun Xiang et al. Front Immunol. .

Abstract

While lymphocytopenia is a common characteristic of coronavirus disease 2019 (COVID-19), the mechanisms responsible for this lymphocyte depletion are unclear. Here, we retrospectively reviewed the clinical and immunological data from 18 fatal COVID-19 cases, results showed that these patients had severe lymphocytopenia, together with high serum levels of inflammatory cytokines (IL-6, IL-8 and IL-10), and elevation of many other mediators in routine laboratory tests, including C-reactive protein, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase and natriuretic peptide type B. The spleens and hilar lymph nodes (LNs) from six additional COVID-19 patients with post-mortem examinations were also collected, histopathologic detection showed that both organs manifested severe tissue damage and lymphocyte apoptosis in these six cases. In situ hybridization assays illustrated that SARS-CoV-2 viral RNA accumulates in these tissues, and transmission electronic microscopy confirmed that coronavirus-like particles were visible in the LNs. SARS-CoV-2 Spike and Nucleocapsid protein (NP) accumulated in the spleens and LNs, and the NP antigen restricted in angiotensin-converting enzyme 2 (ACE2) positive macrophages and dendritic cells (DCs). Furthermore, SARS-CoV-2 triggered the transcription of Il6, Il8 and Il1b genes in infected primary macrophages and DCs in vitro, and SARS-CoV-2-NP+ macrophages and DCs also manifested high levels of IL-6 and IL-1β, which might directly decimate human spleens and LNs and subsequently lead to lymphocytopenia in vivo. Collectively, these results demonstrated that SARS-CoV-2 induced lymphocytopenia by promoting systemic inflammation and direct neutralization in human spleen and LNs.

Keywords: COVID-19; SARS-CoV-2; dendritic cells; lymph nodes; lymphocytopenia; macrophages; spleen.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
SARS-CoV-2 destroys human spleen and LN tissues. The spleens and hilar LNs were collected from one representative fatal COVID-19 (case #2), HBV-ACLF, EBV-infected patients and trauma victim, (A) histopathology was compared by H&E staining, arrow heads show apoptotic cells; (B) cell apoptosis was detected by in situ TUNEL staining (left), with statistical analysis of apoptotic cells (right). Arrows indicate positive cells, *p < 0.05 and **p < 0.01; (C) immunofluorescent staining analysis of Fas expression; (D) immunofluorescent double staining analysis of Fas expression by CD3+ T cells and B220+ B cells in the hilar LNs. Scale bar = 50 μM arrows indicate double positive cells.
Figure 2
Figure 2
SARS-CoV-2 was identified in spleens and LNs. The spleen and hilar LNs from one representative COVID-19 post-mortem case (case #2) were collected, (A) SARS-CoV-2 viral RNA was detected by RNA ISH, the positive reactivity for the housekeeping gene PPIB was used as positive control. Scale bar = 50 μM, arrow indicates positive cells. (B) TEM assay showed that some coronavirus-like particles with diameters of 50~100 nm were observed in hilar LNs. Arrows indicate coronavirus-like particles.
Figure 3
Figure 3
The expression of ACE2 and TMPRSS2 was observed by macrophages and DCs in spleens and hilar LNs. The spleen, hilar LNs and lung tissues from one representative COVID-19 post-mortem case (case #2) were collected, (A) the expression of ACE2 and TMPRSS2 was detected by IHC, arrows indicate positive cells; (B) immunofluorescent double staining analysis of ACE2 antigen on indicated cells within hilar LNs, scale bar= 50 μM, arrows indicated double positive cells and arrow heads showed single positive cells.
Figure 4
Figure 4
SARS-CoV-2 directly infects human macrophages and DCs. Human MDMs and MoDCs were mock infected or infected with SARS-CoV-2 (MOI =1.0) for 2h, after virus absorption, cells were washed and continuously cultured for additional 24h, (A)The expression of endogenous ACE2, TMPRSS2 were evaluated in MDMs, MoDCs and Huh7 cells, **p < 0.01. (B) The expression of ACE2 and SARS-CoV-2 NP antigen was detected by immunofluorescent staining; (C) qPCR analysis of SARS-CoV-2 viral RNA, **p < 0.01. (D) the presence of SARS-CoV-2 viral RNA was detected by RNA ISH, the reactivity for PPIB was used as positive control; The spleens and hilar LNs from one representative COVID-19 post-mortem (case #3) were collected, (E) sections were incubated with primary anti-SARS-NP antibodies (clone ID: 019, rabbit IgG; and ab273434, mouse monoclonal 6H3), anti-SARS-S antibodies (ab273433, mouse monoclonal 1A9), or with mouse or rabbit IgG1 control antibodies, and the expression of viral NP and S antigens were analyzed by IHC, scale bar = 50 μM, arrows indicate positive cells; (F) The hilar LNs were incubated with primary anti-SARS-NP antibodies (clone ID: 019, rabbit IgG) and other mouse derived antibodies, SARS-CoV-2 NP antigen in indicated cells was analyzed by immunofluorescent double staining. Scale bar = 50 μM, arrows indicated double positive cells and arrow heads showed single positive cells.
Figure 5
Figure 5
SARS-CoV-2 triggers IL-1β and IL-6 production by macrophages and DCs. Human MDMs and MoDCs were mock infected or infected with SARS-CoV-2 (MOI= 1.0) for 2h, after virus absorption, cells were washed and continuously cultured for 24h, the transcription of the indicated genes in (A) MDMs and (B) MoDC was detected by qRT-PCR, **p < 0.01. The spleens and LNs from one representative COVID-19 patient post-mortem (case #5) and trauma victim were collected, (C) the expression of IL-6 and IL-1β was analyzed by IHC, scale bar= 50 μM, arrows indicate positive cells; Sections from the hilar LNs were selected to analyze the secretion of (D) IL-6 and (E) IL-1β from the indicated cells by immunofluorescent double staining. Scale bar= 50 μM, arrows indicate double positive cells.
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