We have previously shown that the IAV aggregating protein PB1-F2 can elicit an NLRP3 dependent inflammasome response contributing to IAV disease pathogenesis [11, 12]. Therefore, we hypothesized that other viral aggregating proteins may drive inflammasome activity and contribute to disease inflammation. Through in-silico analysis of other viral proteins, we identified that the C-protein of Hendra virus (HeVc), had high aggregative potential similar to that of IAV PB1-F2. Furthermore, the C-proteins of paramyxoviruses, are expressed in an alternate reading frame and have immunomodulatory effects when in the cytosol of cells, similarly to that previously described for PB1-F2 . Therefore, we sought to identify whether extracellular protein aggregates of Hendra virus C-protein would be effective at inflammasome activation. As Hendra virus is a biosafety level 4 pathogen we derived a peptide from HeVc protein containing the N-terminus portion aggregating potential for further experiments (Fig. 1A), which we visually confirmed formed aggregates in PBS (Supplemental Fig. 1).
HeVc peptide induces IL-1β secretion in murine macrophages through NLRP3 inflammasome activation
To determine whether the HeVc peptide was able to induce IL-1β secretion and possible inflammasome activation, we challenged immortalized murine bone marrow-derived macrophages (iBMDMs) with a range of concentrations of HeVc peptide, or with the well-described NLRP3 inflammasome activators silica and nigericin for 6 h as controls. As can be seen in Fig. 1B, HeVc peptide significantly induced IL-1β secretion in iBMDMs at 200 µg/mL, as was also observed for nigericin and silica respectively. Furthermore, treatment with a scrambled soluble HeVc peptide (Fig. 1C) did not induce significant IL-1β secretion, highlighting the insoluble aggregating nature of HeVc peptide is required for IL-1β secretion.
The phagocytosis of disease- and infection-related protein or peptide aggregates by macrophages, is a well characterized inducer of NLRP3 inflammasome activation [9,10,11]. As can be seen in Fig. 2A, inhibition of phagocytosis with Latrunculin A inhibited HeVc-induced IL-1β secretion, while targeting of caspase-1 catalytic activity with Ac-YVAD-cmk (Fig. 2B) or VX765 (Fig. 2C), similarly significantly reduced IL-1β secretion. Finally, MCC950, a specific small molecule NLRP3 inhibitor inhibited IL-1β secretion (Fig. 2D) commensurate with probenecid (Fig. 2E), which has previously been demonstrated to target aggregated IAV PB1-F2 NLRP3 inflammasome activation [12, 15].
Taken together, these results demonstrate HeVc peptide requires phagocytosis to induce activation of an NLRP3 inflammasome to mediate caspase-1-mediated enzymatic IL-1β secretion in both mouse and human macrophages.
To further determine whether the NLRP3 inflammasome was the major driver of the response to extracellular HeVc peptide, we examined IL-1β secretion from iBMDM cells individually deficient in NLRP3, the adaptor protein ASC or Caspase-1 following aggregated HeVc peptide challenge. Clearly highlighting the requirement of NLRP3, ASC, or caspase-1 for HeVc peptide to activate an inflammasome complex, IL-1β secretion was significantly attenuated in the absence of all NLRP3 inflammasome components (Fig. 3A), compared to wildtype (WT) macrophages. Critically, responses to known NLRP3 agonists Nigericin (Fig. 3C) and silica (Fig. 3B) were also attenuated, while induction of the AIM2 inflammasome with poly (dA:dT), was able to induce IL-1β secretion in NLRP3-deficient cell lines, but not ASC or Caspase-1 deficient iBMDMs (Fig. 3D), suggesting a specificity for NLRP3 activation for HeVc peptide. Together, these results identify that aggregated HeVc peptide induces the activation of an NLRP3 inflammasome complex that leads to IL-1β secretion from macrophages.
HeVc peptide induces ASC-speck formation in ASC-cerulean cell lines
As we had determined that HeVc peptide required NLRP3 inflammasome activation for eliciting IL-1β secretion in iBMDMs, we next sought to visualize inflammasome activation through ASC-speck formation. NLRP3-deficient iBMDMs reconstituted with ASC-cerulean and NLRP3-Flag were stimulated with HeVc peptide or Nigericin and ASC-speck formation observed using confocal microscopy. As can be observed in Fig. 4A, while no to limited specks were observed in unchallenged iBMDMs, HeVc peptide was able to induce ASC speck formation (Fig. 4A, ASC-cerulean, white arrows) after 5 h of challenge in the cytosol of cells, indicative of formation of an inflammasome complex, similar to that observed for Nigericin (bottom panels). Quantification of cytosolic ASC specks (Fig. 4B) demonstrated that both HeVc peptide and Nigericin, induced significantly increased inflammasome complex formation compared to untreated cells. These observations further highlight NLRP3 inflammasome formation in response to HeVc peptide and expands the role of the NLRP3 inflammasome in IL-1β secretion in response to extracellular HeVc peptide.
HeVc peptide induces IL-1β secretion in human THP-1 derived macrophages and human peripheral blood mononuclear cells that is reduced by NLRP3 inhibition
Given the inflammatory response in humans contributes to disease pathology and mortality, we next sought to determine whether HeVc peptide was able to activate the NLRP3 inflammasome in human macrophages and generate mature IL-1β. Human THP-1 differentiated macrophages were primed with LPS prior to stimulation with a range of aggregated HeVc peptide concentrations. As can be seen in Fig. 5A, HeVc peptide elicited a dose-dependent secretion of IL-1β, which was dose-dependently inhibited by MCC950 treatment (Fig. 5B); similar to our results in murine macrophages (Fig. 2). To confirm that HeVc-induced maturation of caspase-1 and IL-1β, we next verified by immunoblot that HeVc-induced both caspase-1 (Fig. 5C) and IL-1β (Fig. 5D) maturation into their bioactive p20 and p17 forms respectively, which critically, was inhibited with MCC950. These observations would reinforce that aggregated HeVc peptide induces an NLRP3 inflammasome leading to release of bioactive IL-1β in human macrophages. To validate that HeVc peptide may also enhance inflammatory events in human cells, we examined cellular responses in LPS-primed human peripheral blood mononuclear cells (hPBMCs) exposed to HeVc peptide as compared to scrambled peptide. As can be seen in Fig. 5E, while HeVc peptide induced IL-1β secretion from hPBMCs, scrambled peptide did not. Importantly, treatment with MCC950 significantly inhibited this secretion, suggesting HeVc peptide activated the NLRP3 inflammasome.
HeVc peptide induces pulmonary inflammation in an in vivo challenge model
To identify whether HeVc peptide was able to activate the NLRP3 inflammasome in an in vivo setting, we intranasally challenged C57BL/6 mice with HeVc peptide and assessed cellular infiltrates and the secretion of IL-1β through bronchoalveolar lavage (BAL) analysis. Mice challenged with aggregated HeVc peptide for 6 h showed a significant increase in IL-1β secreted into the bronchoalveolar space (Fig. 6A). Critically, intranasal treatment with the NLRP3 inhibitor MCC950  significantly reduced IL-1β in BAL fluid, suggesting that IL-1β secretion in response to HeVc peptide is driven by the NLRP3 inflammasome. Conversely, while IL-6 was increased in response to aggregated peptide challenge, MCC950 did not reduce IL-6 secretion into the BALF, further highlighting the specific nature of the immune response to aggregated peptide via NLRP3. Inflammation in the lung was further highlighted by a significant increase of neutrophils in the bronchoalveolar space (Fig. 6C). Importantly, treatment with MCC950 did not reduce the number of neutrophils and IL-6 (Fig. 6B-C), highlighting its specificity in inhibiting the NLRP3 inflammasome and not due to reduced cellular infiltrates due to decreased IL-1β secretion. Consistent with this, total lung cell counts (Fig. 6D) were unchanged between all groups. Taken together, these data highlight the propensity for HeVc peptide, and thus Hendra virus C-protein, to initiate an inflammatory response in-vivo through the activation of the NLRP3 inflammasome. Critically, we also demonstrate that IL-1β secretion into the pulmonary space can be inhibited with NLRP3 inhibitors, highlighting the possibility of targeting NLRP3 activity to reduce pulmonary inflammation during Hendra Virus infection.