Host Pattern Recognition Receptors Research Articles

Role of the nucleotide-binding oligomerization domain-containing protein 1 pathway in the development of periodontitis

ObjectivesDuring innate immune defense, host pattern recognition receptors, including toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), can activate downstream pathways by recognizing pathogen-associated molecular patterns produced by microorganisms, triggering immune responses. NOD1, an important cell membrane protein in the NLR-like receptor protein family, exerts anti-infective effects through γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) recognition. Oral epithelial cells resist bacterial invasion through iE-DAP-induced interleukin (IL)-8 production, recruiting neutrophils to sites of inflammation in response to bacterial threats to periodontal tissues. To date, the regulatory mechanisms of iE-DAP in gingival epithelial cells (GECs) are poorly understood. This study was conducted to investigate the role of the NOD1 pathway in the development of periodontitis by examining the effect of iE-DAP on IL-8 production in Ca9-22 cells. MethodsIL-8 production by iE-DAP-stimulated-Ca9-22 cells was assessed using an enzyme-linked immunosorbent assay. Phosphorylation levels of intracellular signaling molecules were evaluated using western blot analyses.Results: iE-DAP induced NOD1 receptor expression in Ca9-22 cells. Additionally, iE-DAP induced expression of pro-IL-1β protein without extracellular secretion. Our results suggest that iE-DAP regulates IL-8 production by activating p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signaling pathways. iE-DAP also promoted nuclear factor kappa-B p65 phosphorylation, facilitating its nuclear translocation. Notably, p38 MAPK and ERK1/2 inhibitors suppressed iE-DAP-stimulated IL-8 production, suggesting that JNK is not involved in this mechanism. ConclusionsOur results indicate that p38 MAPK and ERK1/2, but not JNK, are involved in innate immune responses in GECs.

Effect of Recombinant Glutathione-S-Transferase A3 Protein on the Expression of Host Defense Resistance and Pattern Recognition Receptor in the Thiram Induced Tibial Dyschondroplasia Chicken Erythrocytes

Effect of Recombinant Glutathione-S-Transferase A3 Protein on the Expression of Host Defense Resistance and Pattern Recognition Receptor in the Thiram Induced Tibial Dyschondroplasia Chicken Erythrocytes

Hepatitis C virus NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes.

During the replication of viral genomes, RNA viruses produce double-stranded RNA (dsRNA), through the activity of their RNA-dependent RNA polymerases (RdRps) as viral replication intermediates. Recognition of viral dsRNA by host pattern recognition receptors - such as retinoic acid-induced gene-I (RIG-I)-like receptors and Toll-like receptor 3 - triggers the production of interferon (IFN)-β via the activation of IFN regulatory factor (IRF)-3. It has been proposed that, during the replication of viral genomes, each of RIG-I and melanoma differentiation-associated gene 5 (MDA5) form homodimers for the efficient activation of a downstream signalling pathway in host cells. We previously reported that, in the non-neoplastic human hepatocyte line PH5CH8, the RdRp NS5B derived from hepatitis C virus (HCV) could induce IFN-β expression by its RdRp activity without the actual replication of viral genomes. However, the exact mechanism by which HCV NS5B produced IFN-β remained unknown. In the present study, we first showed that NS5B derived from another Flaviviridae family member, GB virus B (GBV-B), also possessed the ability to induce IFN-β in PH5CH8 cells. Similarly, HCV NS5B, but not its G317V mutant, which lacks RdRp activity, induced the dimerization of MDA5 and subsequently the activation of IRF-3. Interestingly, immunofluorescence analysis showed that HCV NS5B produced dsRNA. Like HCV NS5B, GBV-B NS5B also triggered the production of dsRNA and subsequently the dimerization of MDA5. Taken together, our results show that HCV NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes in human hepatocytes.

Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation.

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.

Functional immune boosters; the herb or its dead microbiome? Antigenic TLR4 agonist MAMPs found in 65 medicinal roots and algae's.

Humans have been consuming medicinal plants (as herbs/ spices) to combat illness for centuries while ascribing beneficial effects predominantly to the plant/phytochemical constituents, without recognizing the power of obligatory resident microorganism' communities (MOCs) (live/dead bacteria, fungus, yeast, molds etc.) which remain after industrial microbial reduction methods. Very little is known about the taxonomic identity of residual antigenic microbial associated molecular patterns (MAMPs) debris in our botanical over the counter (OTC) products, which if present would be recognized as foreign (non-self) antigenic matter by host pattern recognition receptors (PRRs) provoking a host immune response; this the basis of vaccine adjuvants. As of today, only few research groups have removed the herbal MAMP biomass from herbs, all suggesting that immune activation may not be from the plant but rather its microbial biomass; a hypothesis we corroborate. The purpose of this work was to conduct a high through put screening (HTPS) of over 2500 natural plants, OTC botanical supplements and phytochemicals to elucidate those with pro-inflammatory; toll like receptor 4 (TLR4) activating properties in macrophages. The HTPS was conducted on RAW 264.7 cells vs. lipopolysaccharide (LPS) E. coli 0111:B4, testing iNOS / nitric oxide production as a perimeter endpoint. The data show not a single drug/chemical/ phytochemical and approximately 98 % of botanicals to be immune idle (not effective) with only 65 pro-inflammatory (hits) in a potency range of LPS. Method validation studies eliminated the possibility of false artifact or contamination, and results were cross verified through multiple vendors/ manufacturers/lot numbers by botanical species. Lead botanicals were evaluated for plant concentration of LPS, 1,3:1,6-β-glucan, 1,3:1,4-β-D-glucan and α-glucans; where the former paralleled strength in vitro. LPS was then removed from plants using high-capacity endotoxin poly lysine columns, where bioactivity of LPS null "plant" extracts were lost. The stability of E.Coli 0111:B4 in an acid stomach mimetic model was confirmed. Last, we conducted a reverse culture on aerobic plate counts (APCs) from select hits, with subsequent isolation of gram-negative bacteria (MacConkey agar). Cultures were 1) heat destroyed (retested/ confirming bioactivity) and 2) subject to taxonomical identification by genetic sequencing 18S, ITS1, 5.8 s, ITS2 28S, and 16S. The data show significant gram negative MAMP biomass dominance in A) roots (e.g. echinacea, yucca, burdock, stinging nettle, sarsaparilla, hydrangea, poke, madder, calamus, rhaponticum, pleurisy, aconite etc.) and B) oceanic plants / algae's (e.g. bladderwrack, chlorella, spirulina, kelp, and "OTC Seamoss-blends" (irish moss, bladderwrack, burdock root etc), as well as other random herbs (eg. corn silk, cleavers, watercress, cardamom seed, tribulus, duckweed, puffball, hordeum and pollen). The results show a dominance of gram negative microbes (e.g. Klebsilla aerogenes, Pantoae agglomerans, Cronobacter sakazakii), fungus (Glomeracaea, Ascomycota, Irpex lacteus, Aureobasidium pullulans, Fibroporia albicans, Chlorociboria clavula, Aspergillus_sp JUC-2), with black walnut hull, echinacea and burdock root also containing gram positive microbial strains (Fontibacillus, Paenibacillus, Enterococcus gallinarum, Bromate-reducing bacterium B6 and various strains of Clostridium). This work brings attention to the existence of a functional immune bioactive herbal microbiome, independent from the plant. There is need to further this avenue of research, which should be carried out with consideration as to both positive or negative consequences arising from daily consumption of botanicals highly laden with bioactive MAMPS.

Mitogen-Activated Protein Kinases (MAPKs) and Enteric Bacterial Pathogens: A Complex Interplay.

Diverse extracellular and intracellular cues activate mammalian mitogen-activated protein kinases (MAPKs). Canonically, the activation starts at cell surface receptors and continues via intracellular MAPK components, acting in the host cell nucleus as activators of transcriptional programs to regulate various cellular activities, including proinflammatory responses against bacterial pathogens. For instance, binding host pattern recognition receptors (PRRs) on the surface of intestinal epithelial cells to bacterial pathogen external components trigger the MAPK/NF-κB signaling cascade, eliciting cytokine production. This results in an innate immune response that can eliminate the bacterial pathogen. However, enteric bacterial pathogens evolved sophisticated mechanisms that interfere with such a response by delivering virulent proteins, termed effectors, and toxins into the host cells. These proteins act in numerous ways to inactivate or activate critical components of the MAPK signaling cascades and innate immunity. The consequence of such activities could lead to successful bacterial colonization, dissemination, and pathogenicity. This article will review enteric bacterial pathogens' strategies to modulate MAPKs and host responses. It will also discuss findings attempting to develop anti-microbial treatments by targeting MAPKs.

The IFN-stimulated gene IFI27 counteracts innate immune responses after viral infections by interfering with RIG-I signaling.

The recognition of viral nucleic acids by host pattern recognition receptors (PRRs) is critical for initiating innate immune responses against viral infections. These innate immune responses are mediated by the induction of interferons (IFNs), IFN-stimulated genes (ISGs) and pro-inflammatory cytokines. However, regulatory mechanisms are critical to avoid excessive or long-lasting innate immune responses that may cause detrimental hyperinflammation. Here, we identified a novel regulatory function of the ISG, IFN alpha inducible protein 27 (IFI27) in counteracting the innate immune responses triggered by cytoplasmic RNA recognition and binding. Our model systems included three unrelated viral infections caused by Influenza A virus (IAV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and Sendai virus (SeV), and transfection with an analog of double-stranded (ds) RNA. Furthermore, we found that IFI27 has a positive effect on IAV and SARS-CoV-2 replication, most likely due to its ability to counteract host-induced antiviral responses, including in vivo. We also show that IFI27 interacts with nucleic acids and PRR retinoic acid-inducible gene I (RIG-I), being the interaction of IFI27 with RIG-I most likely mediated through RNA binding. Interestingly, our results indicate that interaction of IFI27 with RIG-I impairs RIG-I activation, providing a molecular mechanism for the effect of IFI27 on modulating innate immune responses. Our study identifies a molecular mechanism that may explain the effect of IFI27 in counterbalancing innate immune responses to RNA viral infections and preventing excessive innate immune responses. Therefore, this study will have important implications in drug design to control viral infections and viral-induced pathology.

Research progress on the effect of traditional Chinese medicine on the activation of PRRs-mediated NF-κB signaling pathway to inhibit influenza pneumonia.

Influenza pneumonia has challenged public health and social development. One of the hallmarks of severe influenza pneumonia is overproduction of pro-inflammatory cytokines and chemokines, which result from the continuous activation of intracellular signaling pathways, such as the NF-κB pathway, mediated by the interplay between viruses and host pattern recognition receptors (PRRs). It has been reported that traditional Chinese medicines (TCMs) can not only inhibit viral replication and inflammatory responses but also affect the expression of key components of PRRs and NF-κB signaling pathways. However, whether the antiviral and anti-inflammatory roles of TCM are related with its effects on NF-κB signaling pathway activated by PRRs remains unclear. Here, we reviewed the mechanism of PRRs-mediated activation of NF-κB signaling pathway following influenza virus infection and summarized the influence of anti-influenza TCMs on inflammatory responses and the PRRs/NF-κB signaling pathway, so as to provide better understanding of the mode of action of TCMs in the treatment of influenza pneumonia.

Current progress on innate immune evasion mediated by Npro protein of pestiviruses.

Interferon (IFN), the most effective antiviral cytokine, is involved in innate and adaptive immune responses and is essential to the host defense against virus invasion. Once the host was infected by pathogens, the pathogen-associated molecular patterns (PAMPs) were recognized by the host pattern recognition receptors (PRRs), which activates interferon regulatory transcription factors (IRFs) and nuclear factor-kappa B (NF-κB) signal transduction pathway to induce IFN expression. Pathogens have acquired many strategies to escape the IFN-mediated antiviral immune response. Pestiviruses cause massive economic losses in the livestock industry worldwide every year. The immune escape strategies acquired by pestiviruses during evolution are among the major difficulties in its control. Previous experiments indicated that Erns, as an envelope glycoprotein unique to pestiviruses with RNase activity, could cleave viral ss- and dsRNAs, therefore inhibiting the host IFN production induced by viral ss- and dsRNAs. In contrast, Npro, the other envelope glycoprotein unique to pestiviruses, mainly stimulates the degradation of transcription factor IRF-3 to confront the IFN response. This review mainly summarized the current progress on mechanisms mediated by Npro of pestiviruses to antagonize IFN production.

Interactions among deep-sea mussels and their epibiotic and endosymbiotic chemoautotrophic bacteria: Insights from multi-omics analysis.

Endosymbiosis with Gammaproteobacteria is fundamental for the success of bathymodioline mussels in deep-sea chemosynthesis-based ecosystems. However, the recent discovery of Campylobacteria on the gill surfaces of these mussels suggests that these host-bacterial relationships may be more complex than previously thought. Using the cold-seep mussel ( Gigantidas haimaensis) as a model, we explored this host-bacterial system by assembling the host transcriptome and genomes of its epibiotic Campylobacteria and endosymbiotic Gammaproteobacteria and quantifying their gene and protein expression levels. We found that the epibiont applies a sulfur oxidizing (SOX) multienzyme complex with the acquisition of soxB from Gammaproteobacteria for energy production and switched from a reductive tricarboxylic acid (rTCA) cycle to a Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. The host provides metabolic intermediates, inorganic carbon, and thiosulfate to satisfy the materials and energy requirements of the epibiont, but whether the epibiont benefits the host is unclear. The endosymbiont adopts methane oxidation and the ribulose monophosphate pathway (RuMP) for energy production, providing the major source of energy for itself and the host. The host obtains most of its nutrients, such as lysine, glutamine, valine, isoleucine, leucine, histidine, and folate, from the endosymbiont. In addition, host pattern recognition receptors, including toll-like receptors, peptidoglycan recognition proteins, and C-type lectins, may participate in bacterial infection, maintenance, and population regulation. Overall, this study provides insights into the complex host-bacterial relationships that have enabled mussels and bacteria to thrive in deep-sea chemosynthetic ecosystems.

Biochemical and Microscopic Analyses for Sphingolipids and Its Related Molecules in Phagosomes.

Glycosphingolipids (GSLs) form GSL-enriched microdomains, together with sphingomyelin (SM), cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and membrane-associated signaling molecules. GSL-enriched microdomains mediate a variety of physiological functions, including innate immune responses. Innate immune responses are initialized by the binding of host pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) expressed in microorganisms. This binding triggers phagocytosis and leads to the formation of a phagosome-containing microorganism and the subsequent lysosomal fusion with a phagosome. To detect the molecular interaction between GSL-enriched microdomains, sphingolipids, and signaling molecules from the uptake of the microorganism until the phagosome-containing microorganism fuses with lysosomes, biochemical and microscopic approaches are indispensable. Here, we describe the detailed methods for isolating phagosomes and observing the molecular interaction using a superresolution microscope. Our methodology provides a strategy for exploring the molecular interaction between the host and pathogen and for developing new treatment approaches.

Viruses utilize ubiquitination systems to escape TLR/RLR-mediated innate immunity.

When the viruses invade the body, they will be recognized by the host pattern recognition receptors (PRRs) such as Toll like receptor (TLR) or retinoic acid-induced gene-I like receptor (RLR), thus causing the activation of downstream antiviral signals to resist the virus invasion. The cross action between ubiquitination and proteins in these signal cascades enhances the antiviral signal. On the contrary, more and more viruses have also been found to use the ubiquitination system to inhibit TLR/RLR mediated innate immunity. Therefore, this review summarizes how the ubiquitination system plays a regulatory role in TLR/RLR mediated innate immunity, and how viruses use the ubiquitination system to complete immune escape.

Differential activation of NLRP3 inflammasome by Acinetobacter baumannii strains.

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1β and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host's innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death in primary macrophages, epithelial lung cell line and mice. We found a variation in survival outcomes of mice and bacterial dissemination in organs among three commercially available A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found variability among A. baumannii strains in activation of the NLRP3 inflammasome, non-canonical Caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokine IL-1β and programmed cell death. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with different virulence to investigate the innate immune response to A. baumannii infection.

[Advances in Research on the Role of Bacterial c-di-AMP in Host Immunity].

Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is a newly-discovered second messenger in bacteria and archaea. By directly binding to or affecting the expression of target proteins, c-di-AMP regulates the physiological functions of bacteria, including maintaining osmotic pressure, balancing central metabolism, monitoring DNA damage, and controlling biofilm and spore formation. As a new pathogen-associated molecular pattern (PAMP), it binds to the host pattern recognition receptor (PRR), induces cyclic GMP-AMP synthase (cGAS)-STING signal axis to produce type Ⅰ interferon by activating the stimulator of interferon genes (STING), and promotes the secretion of inflammatory factors through nuclear factor κB (NF-κB) signaling pathway, thereby playing an important role in host immunity to bacterial infection and tumorigenesis. Due to its immunogenicity, c-di-AMP could be used as an immune adjuvant to provide new targets for the development of vaccines. However, the specific mechanism of action of c-di-AMP in host immunity awaits further exploration. Herein, we presented the structure and biological characteristics of c-di-AMP, and summarized the possible mechanism of c-di-AMP's regulation of host immune response. In addition, we also reported the latest findings on using c-di-AMP as an immune adjuvant in clinical treatment. Research on the function of c-di-AMP and its mechanism of action on host immune response provides new ideas for finding clinical solutions to bacterial resistance, infection control, tumor prevention, and vaccine development in the future.

RARE PRESENTATION OF INVASIVE PULMONARY ASPERGILLOSIS CO-EXISTENCE WITH EHRLICHIOSIS

SESSION TITLE: Pulmonary Involvement in Critical Care Case PostersSESSION TYPE: Case Report PostersPRESENTED ON: 10/17/2022 12:15 pm - 01:15 pmINTRODUCTION: Invasive Pulmonary Aspergillosis (IPA) is a well-documented condition in critically ill patients, but rarely reported as co-existence with Ehrlichiosis. Our patient presented with Human Monocytic Ehrlichiosis and subsequently developed tracheobronchial aspergillosis. Patient had no predisposing immunosuppressing conditions or risk factors except for IV drug abuse history.CASE PRESENTATION: 48-year-old female with prior history of CHFpEF and polysubstance abuse presented with fever, abdominal pain, nonproductive cough, photophobia, generalized weakness and found to be hypotensive on arrival to hospital. Pertinent laboratory values suggestive of leukopenia, thrombocytopenia, renal azotemia, and transaminitis. Patient was negative for HIV, viral hepatitis, or other immune compromising infections upon initial testing. CT of the chest demonstrated small ground-glass density in the left upper lobe and nonspecific mediastinal/hilar adenopathy. Due to her clinical presentation, patient underwent broad laboratory testing and was positive for E. chaffeensis. However patient continued to deteriorate despite treatment with broad spectrum antibiotics. Diagnostic bronchoscopy performed which demonstrated yellow colored vesicular lesions located diffusely throughout the tracheobronchial trees. Endobronchial biopsy revealed extensive fungal elements in form of fungal balls and fragments, exhibiting septate hyphae with 45-degree branching, consistent with Aspergillosis. Aspergillus galactomannan assay tested positive. In spite of initiating antifungals patient continued to decline and could not survive.DISCUSSION: Ehrlichia utilizes many immune evasion mechanisms, including suppressing apoptosis of host cells, modulation of chemokine and cytokine responses, and down-regulation of host pattern recognition receptors that might enable clearance of the infection, leading to immunocompromised condition. Secondary infections, including those caused by cytomegalovirus, Candida, and Aspergillus spp., have occurred in some severely ill patients. IPA can be challenging to diagnose early due to non-specific presentation. Several studies have demonstrated invasive aspergillosis prevalence in critically ill patients between 0.33-6.9%. Although the incidence rate is low, timely initiation of empiric antifungal therapy is crucial prior to culture results due to high mortality risk of >80%. Ideally tissue biopsy is used to confirm the diagnosis however if it is not feasible, fungal serum labs may be used in conjunction with bronchoalveolar lavage culture. In suspected patients early bronchoscopy can help to confirm aspergillosis diagnosis early and start antifungals.CONCLUSIONS: It is unclear if Ehrlichiosis induced immunosuppression lead to IPA. While not often reported together these infections can cause severe morbidity and mortality. We recommend diagnostic bronchoscopy to diagnose this condition early.Reference #1: Dumler, J. S., W. L. Sutker, and D. H. Walker. 1993. Persistent infection with Ehrlichia chaffeensis. Clin. Infect. Dis. 17:903-905.Reference #2: Wouter Meersseman, Katrien Lagrou, Johan Maertens, Eric Van Wijngaerden, Invasive Aspergillosis in the Intensive Care Unit, Clinical Infectious Diseases, Volume 45, Issue 2, 15 July 2007, Pages 205–216, https://doi.org/10.1086/518852Reference #3: Moreno-González G, Ricart de Mesones A, Tazi-Mezalek R, Marron-Moya MT, Rosell A, Mañez R. Invasive Pulmonary Aspergillosis with Disseminated Infection in Immunocompetent Patient. Can Respir J. 2016;2016:7984032. doi: 10.1155/2016/7984032. Epub 2016 May 5. PMID: 27445566; PMCID: PMC4904540.DISCLOSURES: No relevant relationships by Rajinish JesudossNo relevant relationships by Evan PriceNo relevant relationships by Rakesh Vadde SESSION TITLE: Pulmonary Involvement in Critical Care Case Posters SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Invasive Pulmonary Aspergillosis (IPA) is a well-documented condition in critically ill patients, but rarely reported as co-existence with Ehrlichiosis. Our patient presented with Human Monocytic Ehrlichiosis and subsequently developed tracheobronchial aspergillosis. Patient had no predisposing immunosuppressing conditions or risk factors except for IV drug abuse history. CASE PRESENTATION: 48-year-old female with prior history of CHFpEF and polysubstance abuse presented with fever, abdominal pain, nonproductive cough, photophobia, generalized weakness and found to be hypotensive on arrival to hospital. Pertinent laboratory values suggestive of leukopenia, thrombocytopenia, renal azotemia, and transaminitis. Patient was negative for HIV, viral hepatitis, or other immune compromising infections upon initial testing. CT of the chest demonstrated small ground-glass density in the left upper lobe and nonspecific mediastinal/hilar adenopathy. Due to her clinical presentation, patient underwent broad laboratory testing and was positive for E. chaffeensis. However patient continued to deteriorate despite treatment with broad spectrum antibiotics. Diagnostic bronchoscopy performed which demonstrated yellow colored vesicular lesions located diffusely throughout the tracheobronchial trees. Endobronchial biopsy revealed extensive fungal elements in form of fungal balls and fragments, exhibiting septate hyphae with 45-degree branching, consistent with Aspergillosis. Aspergillus galactomannan assay tested positive. In spite of initiating antifungals patient continued to decline and could not survive. DISCUSSION: Ehrlichia utilizes many immune evasion mechanisms, including suppressing apoptosis of host cells, modulation of chemokine and cytokine responses, and down-regulation of host pattern recognition receptors that might enable clearance of the infection, leading to immunocompromised condition. Secondary infections, including those caused by cytomegalovirus, Candida, and Aspergillus spp., have occurred in some severely ill patients. IPA can be challenging to diagnose early due to non-specific presentation. Several studies have demonstrated invasive aspergillosis prevalence in critically ill patients between 0.33-6.9%. Although the incidence rate is low, timely initiation of empiric antifungal therapy is crucial prior to culture results due to high mortality risk of >80%. Ideally tissue biopsy is used to confirm the diagnosis however if it is not feasible, fungal serum labs may be used in conjunction with bronchoalveolar lavage culture. In suspected patients early bronchoscopy can help to confirm aspergillosis diagnosis early and start antifungals. CONCLUSIONS: It is unclear if Ehrlichiosis induced immunosuppression lead to IPA. While not often reported together these infections can cause severe morbidity and mortality. We recommend diagnostic bronchoscopy to diagnose this condition early. Reference #1: Dumler, J. S., W. L. Sutker, and D. H. Walker. 1993. Persistent infection with Ehrlichia chaffeensis. Clin. Infect. Dis. 17:903-905. Reference #2: Wouter Meersseman, Katrien Lagrou, Johan Maertens, Eric Van Wijngaerden, Invasive Aspergillosis in the Intensive Care Unit, Clinical Infectious Diseases, Volume 45, Issue 2, 15 July 2007, Pages 205–216, https://doi.org/10.1086/518852 Reference #3: Moreno-González G, Ricart de Mesones A, Tazi-Mezalek R, Marron-Moya MT, Rosell A, Mañez R. Invasive Pulmonary Aspergillosis with Disseminated Infection in Immunocompetent Patient. Can Respir J. 2016;2016:7984032. doi: 10.1155/2016/7984032. Epub 2016 May 5. PMID: 27445566; PMCID: PMC4904540. DISCLOSURES: No relevant relationships by Rajinish Jesudoss No relevant relationships by Evan Price No relevant relationships by Rakesh Vadde

Knowing me, knowing you: Self and non-self recognition in plant immunity.

Perception of non-self molecules known as microbe-associated molecular patterns (MAMPs) by host pattern recognition receptors (PRRs) activates plant pattern-triggered immunity (PTI). Pathogen infections often trigger the release of modified-self molecules, termed damage- or danger-associated molecular patterns (DAMPs), which modulate MAMP-triggered signaling to shape the frontline of plant immune responses against infections. In the context of advances in identifying MAMPs and DAMPs, cognate receptors, and their signaling, here, we focus on the most recent breakthroughs in understanding the perception and role of non-self and modified-self patterns. We highlight the commonalities and differences of MAMPs from diverse microbes, insects, and parasitic plants, as well as the production and perception of DAMPs upon infections. We discuss the interplay between MAMPs and DAMPs for emerging themes of the mutual potentiation and attenuation of PTI signaling upon MAMP and DAMP perception during infections.

Cek1 regulates ß(1,3)-glucan exposure through calcineurin effectors in Candida albicans.

In order to successfully induce disease, the fungal pathogen Candida albicans regulates exposure of antigens like the cell wall polysaccharide ß(1,3)-glucan to the host immune system. C. albicans covers (masks) ß(1,3)-glucan with a layer of mannosylated glycoproteins, which aids in immune system evasion by acting as a barrier to recognition by host pattern recognition receptors. Consequently, enhanced ß(1,3)-glucan exposure (unmasking) makes fungal cells more visible to host immune cells and facilitates more robust fungal clearance. However, an understanding of how C. albicans regulates its exposure levels of ß(1,3)-glucan is needed to leverage this phenotype. Signal transduction pathways and their corresponding effector genes mediating these changes are only beginning to be defined. Here, we report that the phosphatase calcineurin mediates unmasking of ß(1,3)-glucan in response to inputs from the Cek1 MAPK pathway and in response to caspofungin exposure. In contrast, calcineurin reduces ß-glucan exposure in response to high levels of extracellular calcium. Thus, depending on the input, calcineurin acts as a switchboard to regulate ß(1,3)-glucan exposure levels. By leveraging these differential ß(1,3)-glucan exposure phenotypes, we identified two novel effector genes in the calcineurin regulon, FGR41 and C1_11990W_A, that encode putative cell wall proteins and mediate masking/unmasking. Loss of either effector caused unmasking and attenuated virulence during systemic infection in mice. Furthermore, immunosuppression restored the colonization decrease seen in mice infected with the fgr41Δ/Δ mutant to wild-type levels, demonstrating a reliance on the host immune system for virulence attenuation. Thus, calcineurin and its downstream regulon are general regulators of unmasking.

Roles of RNA Sensors in Host Innate Response to Influenza Virus and Coronavirus Infections

Influenza virus and coronavirus are two important respiratory viruses, which often cause serious respiratory diseases in humans and animals after infection. In recent years, highly pathogenic avian influenza virus (HPAIV) and SARS-CoV-2 have become major pathogens causing respiratory diseases in humans. Thus, an in-depth understanding of the relationship between viral infection and host innate immunity is particularly important to the stipulation of effective control strategies. As the first line of defense against pathogens infection, innate immunity not only acts as a natural physiological barrier, but also eliminates pathogens through the production of interferon (IFN), the formation of inflammasomes, and the production of pro-inflammatory cytokines. In this process, the recognition of viral pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) is the initiation and the most important part of the innate immune response. In this review, we summarize the roles of RNA sensors in the host innate immune response to influenza virus and coronavirus infections in different species, with a particular focus on innate immune recognition of viral nucleic acids in host cells, which will help to develop an effective strategy for the control of respiratory infectious diseases.

Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease.

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is auridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

Chaperoning mechanism of innate immune receptor NOD2 by HSP70

Host pattern‐recognition receptors (PRRs) are the frontline members of the innate immune system. They are responsible for recognizing molecular structures derived from bacteria or damaged cells and producing appropriate innate immune responses against potential threats from pathogenic agents. However, their defective forms can lead to the development of inflammatory diseases. Crohn’s disease (CD), a prevalent and incurable inflammatory bowel disease, has been shown to closely link to three polymorphisms of the intracellular innate receptor nucleotide‐binding oligomerization domain containing 2 (NOD2). Biophysical and biochemical data show that CD‐associated NOD2 mutants have a much shorter half‐life and reduced signaling function compared to the wild‐type, contributing to the downstream chronic inflammation condition found in CD patients. Given NOD2’s involvement in disease development, it has become an attractive target for drug development against CD. Besides the existing effort in developing anti‐inflammatory drugs and immune suppressors, a novel approach focuses on designing peptidomimetics targeting NOD2 based on its positive modulator protein ‐ stress‐induced heat shock protein 70 kDa (HSP70). Overexpression of both full‐length and truncated HSP70 in cells stabilizes NOD2 mutants and rescues their function, indicating that HSP70 can be used as a platform for developing NOD2 peptide agonists. We first determined the direct interactions at a low micromolar affinity between HSP70 and NOD2 by surface plasmon resonance (SPR) and biolayer interferometry (BLI) technologies. The optimized binding assays will further allow screening of potential HSP70‐derived peptides that retain the same affinity for NOD2. To identify the interacting regions on the amino acid level, we will utilize cross‐linking mass spectrometry (XLMS) together with different docking algorithms. The results will give us a glance into the regulatory mechanisms of HSP70 on NOD2 and provide beneficial structural information for structure‐based peptide drug design. In the future, we plan to study the effects of HSP70‐NOD2 interactions on NOD2‐specific immune responses via RNA sequencing and RT‐PCR.