SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs

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.

Machine learning detects SARS-CoV-2 and variants rapidly on DNA aptamer metasurfaces

COVID-19 is detected using reverse transcription polymerase chain reaction (RT-PCR) of nasal swabs. A very sensitive and rapid detection technique using easily-collected fluids like saliva must be developed for safe and precise mass testing. Here, we introduce a metasurface platform for direct sensing of COVID-19 from unprocessed saliva. We computationally screen gold metasurfaces out of a pattern space of 2100 combinations for strongly-enhanced light-virus interaction with machine learning and use it to investigate the presence and concentration of the SARS-CoV-2. We use machine learning to identify the virus from Raman spectra with 95.2% sensitivity and specificity on 36 PCR positive and 33 negative clinical samples and to distinguish wild-type, alpha, and beta variants. Our results could pave the way for effective, safe and quantitative preventive screening and identification of variants.

IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent

Cutaneous group 2 innate lymphoid cells (ILC2) are spatially and epigenetically poised to respond to barrier compromise and associated immunological threats. ILC2, lacking rearranged antigen-specific receptors, are primarily activated by damage-associated cytokines and respond with type 2 cytokine production. To investigate ILC2 potential for direct sensing of skin pathogens and allergens, we performed RNA sequencing of ILC2 derived from in vivo challenged human skin or blood. We detected expression of NOD2 and TLR2 by skin and blood ILC2. Stimulation of ILC2 with TLR2 agonist alone not only induced interleukin-5 (IL-5) and IL-13 expression but also elicited IL-6 expression in combination with Staphylococcus aureus muramyl dipeptide (MDP). Heat-killed skin-resident bacteria provoked an IL-6 profile in ILC2 in vitro that was notably impaired in ILC2 derived from patients with nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations. In addition, we show that NOD2 signaling can stimulate autophagy in ILC2, which was also impaired in patients with NOD2 mutations. Here, we have identified a role for ILC2 NOD2 signaling in the differential regulation of ILC2-derived IL-6 and have reported a previously unrecognized pathway of direct ILC2 bacterial sensing.

CO 2 sensing by connexin26 and its role in the control of breathing

Breathing is essential to provide the O 2 required for metabolism and to remove its inevitable CO 2 by-product. The rate and depth of breathing is controlled to regulate the excretion of CO 2 to maintain the pH of arterial blood at physiological values. A widespread consensus is that chemosensory cells in the carotid body and brainstem measure blood and tissue pH and adjust the rate of breathing to ensure its homeostatic regulation. In this review, I shall consider the evidence that underlies this consensus and highlight historical data indicating that direct sensing of CO 2 also plays a significant role in the regulation of breathing. I shall then review work from my laboratory that provides a molecular mechanism for the direct detection of CO 2 via the gap junction protein connexin26 (Cx26) and demonstrates the contribution of this mechanism to the chemosensory regulation of breathing. As there are many pathological mutations of Cx26 in humans, I shall discuss which of these alter the CO 2 sensitivity of Cx26 and the extent to which these mutations could affect human breathing. I finish by discussing the evolution of the CO 2 sensitivity of Cx26 and its link to the evolution of amniotes.

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

NOD2 modulates immune tolerance via the GM-CSF–dependent generation of CD103+ dendritic cells

Four decades ago, it was identified that muramyl dipeptide (MDP), a peptidoglycan-derived bacterial cell wall component, could display immunosuppressive functions in animals through mechanisms that remain unexplored. We sought to revisit these pioneering observations because mutations in NOD2, the gene encoding the host sensor of MDP, are associated with increased risk of developing the inflammatory bowel disease Crohn’s disease, thus suggesting that the loss of the immunomodulatory functions of NOD2 could contribute to the development of inflammatory disease. Here, we demonstrate that intraperitoneal (i.p.) administration of MDP triggered regulatory T cells and the accumulation of a population of tolerogenic CD103+ dendritic cells (DCs) in the spleen. This was found to occur not through direct sensing of MDP by DCs themselves, but rather via the production of the cytokine GM-CSF, another factor with an established regulatory role in Crohn’s disease pathogenesis. Moreover, we demonstrate that populations of CD103-expressing DCs in the gut lamina propria are enhanced by the activation of NOD2, indicating that MDP sensing plays a critical role in shaping the immune response to intestinal antigens by promoting a tolerogenic environment via manipulation of DC populations.

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally links the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.