A LTB4/CD11b self-amplifying loop drives pyogranuloma formation in chronic granulomatous disease.

Sterile pyogranulomas and heightened cytokine production are hyperinflammatory hallmarks of Chronic Granulomatous Disease (CGD). Using peritoneal cells of zymosan-treated CGD (gp91phox-/-) versus wild-type (WT) mice, an ex vivo system of pyogranuloma formation was developed to determine factors involved in and consequences of recruitment of neutrophils and monocyte-derived macrophages (MoMacs). Whereas WT cells failed to aggregate, CGD cells formed aggregates containing neutrophils initially, and MoMacs recruited secondarily. LTB4 was key, as antagonizing BLT1 blocked neutrophil aggregation, but acted only indirectly on MoMac recruitment. LTB4 upregulated CD11b expression on CGD neutrophils, and the absence/blockade of CD11b inhibited LTB4 production and cell aggregation. Neutrophil-dependent MoMac recruitment was independent of MoMac Nox2 status, BLT1, CCR1, CCR2, CCR5, CXCR2, and CXCR6. As proof of concept, CD11b-deficient CGD mice developed disrupted pyogranulomas with poorly organized neutrophils and diminished recruitment of MoMacs. Importantly, the disruption of cell aggregation and pyogranuloma formation markedly reduced proinflammatory cytokine production.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Introduction: Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. Methods: We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. Results: More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1ß, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. Discussion: These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Heightened turnover and failed maturation of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in chronic granulomatous disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild-type (WT) peritonea were characterized over time after zymosan injection. Although numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206, and CD36, whereas CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNA-sequencing analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs displayed changes in metabolism, adhesion, and reparative functions, whereas CGD MoMacs remained inflammatory. PKH dye labeling revealed that although WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days, with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations seemed to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. In addition, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data show persistent recruitment with fundamental failure of MoMac maturation in CGD.

Plasmacytoid Dendritic Cells Mediate Control of Ross River Virus Infection via a Type I Interferon-Dependent, MAVS-Independent Mechanism.

Ross River virus (RRV) is a mosquito-borne alphavirus that causes epidemics of debilitating musculoskeletal disease. To define the innate immune mechanisms that mediate control of RRV infection, we studied a RRV strain encoding 6 nonsynonymous mutations in nsP1 (RRV-T48-nsP16M) that is attenuated in wild-type (WT) mice and Rag1-/- mice, which are unable to mount adaptive immune responses, but not in mice that lack the capacity to respond to type I interferon (IFN) (Ifnar1-/- mice). Utilizing this attenuated strain, our prior studies revealed that mitochondrial antiviral signaling (MAVS)-dependent production of type I IFN by Ly6Chi monocytes is critical for control of acute RRV infection. Here, we infected Mavs-/- mice with either WT RRV or RRV-T48-nsP16M to elucidate MAVS-independent protective mechanisms. Mavs-/- mice infected with WT RRV developed severe disease and succumbed to infection, whereas those infected with RRV-T48-nsP16M exhibited minimal disease signs. Mavs-/- mice infected with RRV-T48-nsP16M had higher levels of systemic type I IFN than Mavs-/- mice infected with WT virus, and treatment of Mavs-/- mice infected with the attenuated nsP1 mutant virus with an IFNAR1-blocking antibody resulted in a lethal infection. In vitro, type I IFN expression was induced in plasmacytoid dendritic cells (pDCs) cocultured with RRV-infected cells in a MAVS-independent manner, and depletion of pDCs in Mavs-/- mice resulted in increased viral burdens in joint and muscle tissues, suggesting that pDCs are a source of the protective IFN in Mavs-/- mice. These data suggest that pDC production of type I IFN through a MAVS-independent pathway contributes to control of RRV infection.IMPORTANCE Arthritogenic alphaviruses, including Ross River virus (RRV), are human pathogens that cause debilitating acute and chronic musculoskeletal disease and are a significant public health burden. Using an attenuated RRV with enhanced susceptibility to host innate immune responses has revealed key cellular and molecular mechanisms that can mediate control of attenuated RRV infection and that are evaded by more virulent RRV strains. In this study, we found that pDCs contribute to the protective type I interferon response during RRV infection through a mechanism that is independent of the mitochondrial antiviral signaling (MAVS) adaptor protein. These findings highlight a key innate immune mechanism that contributes to control of alphavirus infections.

Discrete viral E2 lysine residues and scavenger receptor MARCO are required for clearance of circulating alphaviruses.

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o'nyong 'nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency.

dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA.

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don't have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

Inflammatory monocytes mediate control of acute alphavirus infection in mice.

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6ChiCCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6Chi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2+ or Gr1+ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR+;Rag1-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of Irf7, and co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was Irf3;Irf7 and Mavs-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in Mavs-/- mice. Finally, reconstitution of Irf3-/-;Irf7-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2+ cells, indicating that CCR2+ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.