key: cord-0427497-2ekrcnmh authors: van der Donk, Lieve E.H.; Eder, Julia; van Hamme, John L.; Brouwer, Philip J.M.; Brinkkemper, Mitch; van Nuenen, Ad C.; van Gils, Marit J.; Sanders, Rogier W.; Kootstra, Neeltje A.; Bermejo-Jambrina, Marta; Geijtenbeek, Teunis B.H. title: SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs date: 2021-09-03 journal: bioRxiv DOI: 10.1101/2021.09.02.458667 sha: 370d8f13bac0fa58891fc15f50a7c9e856e3e94d doc_id: 427497 cord_uid: 2ekrcnmh Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation and potentially multi-organ dysfunction. It remains unclear whether SARS-CoV-2 is sensed by pattern recognition receptors (PRRs) leading to immune activation. Several studies suggest that the Spike (S) protein of SARS-CoV-2 might interact with Toll-like receptor 4 (TLR4) and thereby activate immunity. Here we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor a primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived dendritic cells (DCs) express TLR4 but not ACE2, and DCs were not infected by a primary SARS-CoV-2 isolate. Notably, neither S protein nor the primary SARS-CoV-2 isolate induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs, including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2. Strikingly, infection of ACE2-positive DCs induced type I IFN and cytokine responses, which was inhibited by antibodies against ACE2. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection. Author summary The immune system needs to recognize pathogens such as SARS-CoV-2 to initiate antiviral immunity. Dendritic cells (DCs) are crucial for inducing antiviral immunity and are therefore equipped with both extracellular and intracellular pattern recognition receptors to sense pathogens. However, it is unknown if and how SARS-CoV-2 activates DCs. Recent research suggests that SARS-CoV-2 is sensed by extracellular Toll-like receptor 4 (TLR4). We have previously shown that DCs do not express ACE2, and are therefore not infected by SARS-CoV-2. Here we show that DCs do not become activated by exposure to viral Spike proteins or SARS-CoV-2 virus particles. These findings suggest that TLR4 and other extracellular TLRs do not sense SARS-CoV-2. Next, we expressed ACE2 in DCs and SARS-CoV-2 efficiently infected these ACE2-positive DCs. Notably, infection of ACE2-positive DCs induced an antiviral immune response. Thus, our study suggests that infection of DCs is required for induction of immunity, and thus that intracellular viral sensors rather than extracellular TLRs are important in sensing SARS-CoV-2. Lack of sensing by extracellular TLRs might be an escape mechanism of SARS-CoV-2 and could contribute to the aberrant immune responses observed during COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that causes 55 coronavirus disease 2019 (1). COVID-19 emerged in 2019 in Wuhan, China(2) , and has since 56 spread globally causing a pandemic. The symptoms of COVID-19 vary amongst individuals, ranging 57 from mild respiratory symptoms to severe lung injury, multi-organ dysfunction and death(3-6). Increasing evidence suggests that disease severity depends not solely on viral infection, but also on an 59 excessive host pro-inflammatory response, whereby high concentrations of pro-inflammatory 60 cytokines result in an unfavorable immune response and induce tissue damage (7, 8 (Fig 1A) . The parental 293 cells 93 did not induce IL-8 upon treatment with S protein or S nanoparticle and LPS. These data suggest that 94 S protein of SARS-CoV-2 does not trigger TLR4. Primary monocyte-derived DCs express TLR4 but also other TLRs(21). We therefore exposed primary 96 human DCs to SARS-CoV-2 S nanoparticles and assessed cytokine production by qPCR. Treatment of 97 DCs with S nanoparticles did neither induce type I interferon (IFN) nor cytokines. (Fig 1B-E) . The positive 98 control LPS induced IFNβ ( Fig 1B) and the interferon-stimulated gene (ISG) APOBEC3G (A3G) (Fig 1C) 99 as well as cytokines IL-6 and IL-10 ( Fig 1D, E) . These data strongly suggest that S protein from SARS- (Fig 2A) . TLR4 expression did not affect 108 infection, as infection was comparable between 293/ACE2 and 293/TLR4/ACE2 cells. Next we investigated whether SARS-CoV-2 pseudovirus activates TLR4. SARS-CoV-2 pseudovirus did 110 neither induce IL-8 in parental 293 nor in 293/TLR4 cells ( Fig 2B) . Moreover, ACE2 expression did not 111 induce activation as exposure of ACE2-positive 293 and 293/TLR4 cells to SARS-CoV-2 pseudovirus did 112 not lead to IL-8 production ( Fig 2B) . These data further support the findings that S protein from SARS-113 CoV-2 does not trigger TLR4 and also show that ACE2 does not affect TLR4 signaling. 128 Moreover, SARS-CoV-2 pseudovirus did not induce any cytokines, in contrast to LPS (Fig 3E-H) . These 129 data indicate that the S protein expressed by SARS-CoV-2 pseudovirus does not activate DCs. Next, we exposed DCs to a primary SARS-CoV-2 isolate and determined DC maturation and 131 cytokine production. We have previously shown that DCs do not become infected by primary SARS- transfected DCs were exposed to the primary SARS-CoV-2 isolate for 24h in presence or absence of 146 blocking antibodies against ACE2. ACE2-expressing DCs were infected by SARS-CoV-2 and infection was 147 blocked by antibodies against ACE2 ( Fig 5D) . Notably, infection of DCs with SARS-CoV-2 induced 148 transcription of IFNβ ( Fig 5E) as well as the ISG A3G ( Fig 5F) . 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IL-8 production was determined by ELISA. (B-E) Primary dendritic cells were exposed to LPS or SARS-