Type I Interferon Counters or Promotes Coxiella burnetii Replication Dependent on Tissue.

Coxiella burnetii is an intracellular pathogen and the cause of Q fever. Gamma interferon (IFN-γ) is critical for host protection from infection, but a role for type I IFN in C. burnetii infection has not been determined. Type I IFN supports host protection from a related pathogen, Legionella pneumophila, and we hypothesized that it would be similarly protective in C. burnetii infection. In contrast to our prediction, IFN-α receptor-deficient (IFNAR(-/-)) mice were protected from C. burnetii-induced infection. Therefore, the role of type I IFN in C. burnetii infection was distinct from that in L. pneumophila Mice treated with a double-stranded-RNA mimetic were protected from C. burnetii-induced weight loss through an IFNAR-independent pathway. We next treated mice with recombinant IFN-α (rIFN-α). When rIFN-α was injected by the intraperitoneal route during infection, disease-induced weight loss was exacerbated. Mice that received rIFN-α by this route had dampened interleukin 1ß (IL-1ß) expression in bronchoalveolar lavage fluids. However, when rIFN-α was delivered to the lung, bacterial replication was decreased in all tissues. Thus, the presence of type I IFN in the lung protected from infection, but when delivered to the periphery, type I IFN enhanced disease, potentially by dampening inflammatory cytokines. To better characterize the capacity for type I IFN induction by C. burnetii, we assessed expression of IFN-ß transcripts by human macrophages following stimulation with lipopolysaccharide (LPS) from C. burnetii Understanding innate responses in C. burnetii infection will support the discovery of novel therapies that may be alternative or complementary to the current antibiotic treatment.

Roles of Toll-Like Receptor 2 (TLR2), TLR4, and MyD88 during Pulmonary Coxiella burnetii Infection.

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular, primarily pulmonary, bacterial pathogen. Although much is known about adaptive immune responses against this bacterium, our understanding of innate immune responses against C. burnetii is not well defined, particularly within the target tissue for infection, the lung. Previous studies examined the roles of the innate immune system receptors Toll-like receptor 2 (TLR2) and TLR4 in peripheral infection models and described minimal phenotypes in specific gene deletion animals compared to those of their wild-type controls (S. Meghari et al., Ann N Y Acad Sci 1063:161-166, 2005,http://dx.doi.org/10.1196/annals.1355.025; A. Honstettre et al., J Immunol 172:3695-3703, 2004,http://dx.doi.org/10.4049/jimmunol.172.6.3695) . Here, we assessed the roles for TLR2, TLR4, and MyD88 in pulmonary C. burnetii infection and compared responses to those that occurred in TLR2- and TLR4-deficient animals following peripheral infection. As observed previously, neither TLR2 nor TLR4 was needed for limiting bacterial growth after peripheral infection. In contrast, TLR2 and, to a lesser extent, TLR4 limited growth (or dissemination) of the bacterium in the lung and spleen after pulmonary infection. TLR2, TLR4, and MyD88 were not required for the general inflammatory response in the lungs after pulmonary infection. However, MyD88 signaling was important for infection-induced morbidity. Finally, TLR2 expression on hematopoietic cells was most important for limiting bacterial growth in the lung. These results expand on our knowledge of the roles for TLR2 and TLR4 in C. burnetii infection and suggest various roles for these receptors that are dictated by the site of infection.

Solute carrier 11A1 is expressed by innate lymphocytes and augments their activation.

Solute carrier 11A1 (SLC11A1) is a divalent ion transporter formerly known as the natural resistance-associated macrophage protein (NRAMP1) and the Bcg/Lsh/Ity locus. SLC11A1 was thought to be exclusively expressed in monocyte/macrophages and to have roles in phagosome maturation and cell activation. We characterized the expression of SLC11A1 in the majority of human and bovine γδ T cells and NK cells and in human CD3(+)CD45RO(+) T cells. Consistent with a role for iron-dependent inhibition of protein tyrosine phosphatases, SLC11A1(+) lymphocytes were more prone to activation and retained tyrosine phosphorylation. Transfection of SLC11A1 into a human γδ T cell-like line rendered the cells more prone to activation. Nonadherent splenocytes from wild-type mice expressed significantly greater IFN-γ compared with cells from Sv/129 (SLC11A1(-/-)) mice. Our data suggest that SLC11A1 has a heretofore unknown role in activation of a large subset of innate lymphocytes that are critical sources of IFN-γ. SLC11A1(+) animals have enhanced innate IFN-γ expression in response to Salmonella infection compared with SLC11A1(-) mice, which include commonly used inbred laboratory mice. Expression of SLC11A1 in innate lymphocytes and its role in augmenting their activation may account for inconsistencies in studies of innate lymphocytes in different animal models.

A TLR4 agonist liposome formulation effectively stimulates innate immunity and enhances protection from bacterial infection.

Stimulation of innate immunity can protect against infectious insult and could be used in combination with other therapies. Since antibiotic resistance is an increasing concern, strategies to reduce the dose or eliminate the need for these drugs are warranted. Lipo-CRX is a formulation in which the TLR4 agonist CRX-527 is incorporated into lipid membranes in liposomes. Lipo-CRX is less inflammatory than either CRX-527 or LPS, but retains unique capacity to enhance host defense responses. We compared lipo-CRX to other agonists in vitro using mammalian cells and in vivo in mice, and assessed indicators of innate immune responses and protection from bacterial infection. Lipo-CRX is similar to E. coli LPS in its capacity to activate bovine γδ T cells and to recruit neutrophils into mouse lungs, but with less reactivity in the LAL assay. However, lipo-CRX uniquely induced the production of systemic innate immune cytokines. In the mouse model of brucellosis, delivery of lipo-CRX to the lungs reduced the dissemination of B. abortus. While lipo-CRX or the antibiotic ampicillin alone did not alter B. abortus burdens in the lung, the combination had a synergistic beneficial effect. Our data suggest that stimulating the innate immune system with lipo-CRX, either alone or when combined with antibiotics, can enhance bacterial clearance in the mouse model of brucellosis.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model.

Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM17) is a protease that cleaves ectodomains of transmembrane proteins, including that of ACE2 and the proinflammatory cytokine TNF-α, from cell surfaces upon cellular activation. We hypothesized that blockade of ADAM17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM17 using the monoclonal antibody MEDI3622 in the K18-hACE2 transgenic mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly lower lung pathology than saline-treated mice. However, the viral burden in the lungs of MEDI3622-treated mice was significantly increased. Thus, ADAM17 appears to have a critical anti-viral role, but also may promote inflammatory damage. Since the inflammatory cascade is ultimately the reason for adverse outcomes in COVID-19 patients, there may be a therapeutic application for the MEDI3622 antibody.

Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection, with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly, SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Severe Acute Respiratory Syndrome Coronavirus 2 Is Detected in the Gastrointestinal Tract of Asymptomatic Endoscopy Patients but Is Unlikely to Pose a Significant Risk to Healthcare Personnel.

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

Titers, Prevalence, and Duration of SARS-CoV-2 Antibodies in a Local COVID-19 Outbreak and Following Vaccination.

Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor-binding domain (RBD)-specific antibodies, their duration, and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest-lasting response, up to 12 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression.

Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7aΔ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncations of ORF7a that limit immune suppression.

Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We have isolated one of these mutant viruses from a patient sample and used viral challenge experiments to demonstrate that Δ115 mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response.

Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.

Complete Genome Sequence of Brucella abortus Phage EF4, Determined Using Long-Read Sequencing.

Brucellaphage EF4 was isolated from elk feces. The 38,321-bp double-stranded DNA genome is predicted to contain 72 coding regions, 38 of which have been assigned predicted functions. This phage displays nucleotide similarity to other brucellaphages of the genus Perisivirus.

The distinct response of gammadelta T cells to the Nod2 agonist muramyl dipeptide.

Purified gammadelta T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. Transcripts encoding the innate receptor Nod2 were detected in bovine and human gammadelta T cells. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), functions in regulating innate activities, and was thought to be expressed primarily in APCs. The response of gammadelta T cells to MDP was analyzed by microarray, Q-PCR, proteome array and functional priming assays. MDP had a consistent priming effect on gammadelta T cells, characterized by changes in transcripts and enhanced proliferation response to secondary signaling. Knockdown experiments implicated Nod2 as the receptor for MDP in gammadelta T cell-enriched bovine PBLs. The results indicate priming of gammadelta T cells by MDP, and offer definitive evidence of the expression of functional Nod2 in gammadelta T cells.

Response of gammadelta T Cells to plant-derived tannins.

Many pharmaceutical drugs arc isolated from plants used in traditional medicines, and new plant-derived pharmaceutical drugs continue to be identified. Relevant to this review, different plant-derived agonists for gammadelta T cells are described that impart effector functions upon distinct subsets of these cells. Recently, plant tannins have been defined as one class of gammadelta T cell agonist and appear to preferentially activate the mucosal population. Mucosal gammadelta T cells function to modulate tissue immune responses and induce epithelium repair. Select tannins, isolated from apple peel, rapidly induce immune gene transcription in gammadelta T cells, leading to cytokinc production and increased responsiveness to secondary signals. Activity of these tannin preparations tracks to the procyanidin fraction, with the procyanidin trimer (C1) having the most robust activity defined to date. The response to the procyanidins is evolutionarily conserved in that responses are seen with human, bovine, and murine gammadelta T cells, although human cells show less selectivity. Procyanidin-induced responses described in this review likely account for the expansion of mucosal gammadelta T cells seen in mice and rats fed soluble extracts of tannins. Use of procyanidins to activate gammadelta T cells may represent a novel approach for the treatment of tissue damage and autoimmune diseases.

Antigen-independent priming: a transitional response of bovine gammadelta T-cells to infection.

Analysis of global gene expression in immune cells has provided unique insights into immune system function and response to infection. Recently, we applied microarray and serial analysis of gene expression (SAGE) techniques to the study of gammadelta T-cell function in humans and cattle. The intent of this review is to summarize the knowledge gained since our original comprehensive studies of bovine gammadelta T-cell subsets. More recently, we have characterized the effects of mucosal infection or treatment with microbial products or mitogens on gene expression patterns in sorted gammadelta and alphabeta T-cells. These studies provided new insights into the function of bovine gammadelta T-cells and led to a model in which response to pathogen-associated molecular patterns (PAMPs) induces 'priming' of gammadelta T-cells, resulting in more robust responses to downstream cytokine and/or antigen signals. PAMP primed gammadelta T-cells are defined by up-regulation of a select number of cytokines, including MIP1alpha and MIP1beta, and by antigens such as surface IL2 receptor alpha (IL-2Ralpha) and CD69, in the absence of a prototypic marker for an activated gammadelta T-cell, IFN-gamma. Furthermore, PAMP primed gammadelta T-cells are more capable of proliferation in response to IL-2 or IL-15 in the absence of antigen. PAMPs such as endotoxin, peptidoglycan and beta-glucan are effective gammadelta T-cell priming agents, but the most potent antigen-independent priming agonists defined to date are condensed oligomeric tannins produced by some plants.

Comparative gene expression by WC1+ gammadelta and CD4+ alphabeta T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ gammadelta T cells.

The functions of gammadelta T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized alphabeta T cells. However, their conservation throughout evolution suggests that gammadelta T cells are biologically unique. In ruminants, gammadelta T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from alphabeta T cells. In fact, bovine WC1(+) gammadelta T cells can act as APC for alphabeta T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1(+) gammadelta and CD4(+) alphabeta T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific alphabeta and gammadelta T cell clones express many of the same genes, gammadelta T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1(+) gammadelta T cells when compared with CD4(+) alphabeta T cells selected from peripheral blood.

Distinct gene expression in human Vdelta1 and Vdelta2 gammadelta T cells following non-TCR agonist stimulation.

The two major human gammadelta T cell subsets, Vdelta1 and Vdelta2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vdelta1 and Vdelta2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vdelta2 cells being more inflammatory and Vdelta1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of gammadeltaT cells to the innate immune system. Our results indicate that circulating Vdelta1 and Vdelta2 subsets in humans have considerable inherent differences in gene expression following treatment with the same agonist. The patterns of differentially expressed genes are consistent with unique functional roles for these cells in vivo.

Getting "Inside" Type I IFNs: Type I IFNs in Intracellular Bacterial Infections.

Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial "sensing" mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.