Extracellular Heme Impairs Inflammatory Responses of Mononuclear Phagocytes to TLR7 and TLR 8 Stimulation

IntroductionThe COVID-19 pandemic has had an unprecedented impact with over 73 million confirmed cases and over1.6 million deaths globally at the time of this writing. Patients with severe SARS-CoV-2 infection can develop acute respiratory distress syndrome (ARDS). The illness is associated with tissue hemorrhage, causing local hemolysis and the release of heme in the lung. Extracellular heme is rapidly oxidized from Fe2+ to Fe3+, and has been reported to activate MYD88-mediated inflammatory signaling through Toll-like receptor (TLR)-4. SARS-CoV2 is a single strand RNA virus, which is sensed in part by endosomal TLR7/8 for initiating host innate responses. We hypothesized that heme impairs TLR7/8 mediated type 1 interferon response to coronaviruses, thereby worsening the infection. MethodsPeripheral blood mononuclear cells (PBMC) were isolated from each of 6 donor leukocytes, followed by CD14+ isolation. CD14+ monocytes were either studied directly or further differentiated into macrophages under the stimulation of M-CSF. Resiquimod (R848), an agonist for TLR7/8 in humans and TLR7 in a murine systems, was used as a mimic of coronaviruses. CD14+ monocytes and monocytes derived macrophages (MDM) were treated with hemin (heme containing Fe3+ iron), R848, or a combination of hemin and R848. Untreated cells were used as control. The production of proinflammatory cytokines including TNF, IL6, IFN-alpha, and IFN-beta, were measured by ELISA. The expressions of type 1 interferon stimulated genes (x MX1, ISG15, ISG20, and OASL) were assessed via quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results Hemin alone has no effect on the expression of IL6, TNF, or type 1 interferon-stimulated genes by CD14+ monocytes and MDM, in comparison to untreated cells. In monocytes, hemin resulted in marked inhibition of type-I interferon responses induced by TLR7/8 agonism, but had no effect on IL6 or TNF production. In macrophages, hemin significantly downregulated the production of IL6, but did not affect TNF production or interferon signaling induced by TLR7/8 agonism. Conclusion Heme appears to impair the type 1 interferon response to coronavirus and proinflammatory cytokines productions in human mononuclear phagocytes. Furthermore, heme has different effects on human primary monocytes as compared to macrophages in vitro. We speculate that heme may thus worsens the infection caused by SARS-CoV2 in vivo. Supported by: NIH R01AI135128 and U01EB024501.