Silvia M. Uriarte

This work defines multiple roles of mature neutrophils in regulation of the immune response to, and the induction of acute lung injury after, SARS-CoV-2 infection. This research defines neutrophil exocytosis as a therapeutic target to prevent lung injury following SARS-CoV-2 infection.

Introduction: Dysregulated neutrophil functions play a crucial role in the onset and progression of periodontitis. Peptoanaerobacter stomatis is an emerging oral bacterium associated with periodontitis and is known to activate neutrophils through TLR2/6, leading to ROS production, granule release, and neutrophil extracellular trap (NET) formation. In this study, we characterized MAPK kinases that regulate these neutrophil responses and determined the mechanism responsible for bacterial killing. Methods: Human neutrophils were challenged with P. stomatis, and kinase activation was assessed by immunoblot. Functional relevance was tested using selective kinase inhibitors prior to bacterial exposure. Neutrophil responses measured included ROS production by kinetic fluorometric assay, degranulation by flow cytometry, and NET formation using immunofluorescence and immunoblotting. Bacterial killing was determined by a CFU assay. Results: P. stomatis activated ERK and p38 MAPKs, PI3K/AKT, and Src and Syk tyrosine kinases in neutrophils. Inhibition of all kinases except AKT significantly reduced ROS production. Because TAK1 is an upstream regulator of MAPK signaling, its role was also evaluated. Degranulation was mainly regulated by TAK1 and p38, with partial involvement of ERK and Src. NET formation required TAK1, ROS, and PAD4. TAK1 and degranulation inhibition markedly decreased bacterial killing, whereas inhibition of ROS or NET had no effect. Conclusion: These findings demonstrate that although P. stomatis evades ROS- and NET-mediated killing, it remains susceptible to TAK1-mediated neutrophil degranulation, identifying degranulation as the primary mechanism for bacterial clearance.

Endogenous pathogens can constrain virulence to ensure survival in the host. Pathogenic state can be controlled by metabolic responses to the prevailing microenvironment; however, the coupling and effector mechanisms are not well understood. Flux through the one-carbon metabolism (OCM) pathway can modulate virulence of the oral pathobiont Porphyromonas gingivalis , and here we show that this is controlled by tyrosine phosphorylation-dependent differential partitioning of gingipain proteases. The OCM essential precursor pABA inhibits the low molecular weight tyrosine phosphatase Ltp1, and consequently relieves inhibition of its cognate kinase, Ptk1. We found that in the absence of pABA, reduced Ptk1 kinase activity blocks extracellular release of gingipains. Surface retention of gingipains confers resistance to neutrophil mobilization and killing, and virulence in animal models of disease is elevated. Reciprocally, Ptk1 and gingipains are required for maximal flux through OCM, and Ptk1 can phosphorylate the OCM pathway enzymes GlyA and GcvT. Further, ALP, an alkaline phosphatase involved in synthesis of DHPPP, which combines with pABA to make DHP, is phosphorylated and activated by Ptk1. We propose, therefore, that although the primary function of Ptk1 is to maintain OCM balance, it mechanistically couples metabolism with tunable pathogenic potential through directing the location of proteolytic virulence factors.

Enrichment of the oral microorganism Filifactor alocis is strongly associated with the progression of periodontitis. However, F. alocis is part of a complex dysbiotic microbial community, and the organism’s direct pathogenic potential remains uncharacterized. Using the oral gavage model of experimental periodontitis, we revealed that F. alocis promotes alveolar bone loss, as well as significant overexpression of proinflammatory markers associated with osteoclastogenesis and inflammation in oral tissues. Interestingly, despite colonizing in low abundance, F. alocis infection promoted the perturbation of the homeostatic oral microbial community toward a dysbiotic state. Systemically, high levels of proinflammatory cytokines, chemokines, and antibody titers against F. alocis were detected in the sera of infected animals. Germ-free mice or TLR2-deficient mice did not develop alveolar bone loss when infected with F. alocis , demonstrating that the commensal microbial community and the presence of TLR2 are required for F. alocis to display its pathogenic potential. These findings identify F. alocis as an oral pathogen that can disrupt the homeostatic relationship between the host and the oral microbiome, causing inflammation and alveolar bone resorption. Our findings provide novel insights into the pathogenic potential of F. alocis in periodontitis.

Yersinia pestis is the etiologic agent of the plague. A hallmark of plague is subversion of the host immune response by disrupting host signalling pathways required for inflammation. This non‐inflammatory environment permits bacterial colonization and has been shown to be essential for disease manifestation. Previous work has shown that Y. pestis inhibits phagocytosis and degranulation by neutrophils. Manipulation of these key vesicular trafficking pathways suggests that Y. pestis influences extracellular vesicle (EV) secretion, cargo selection, trafficking and/or maturation. Our goals were to define the EV population produced by neutrophils in response to Y. pestis and determine how these vesicles might influence inflammation. Towards these goals, EVs were isolated from human neutrophils infected with Y. pestis or a mutant lacking bacterial effector proteins known to manipulate host cell signalling. Mass spectrometry data revealed that cargoes packaged in EVs isolated from mutant infected cells were enriched with antimicrobial and cytotoxic proteins, contents which differed from uninfected and Y. pestis infected cells. Further, EVs produced in response to Y. pestis lacked inflammatory properties observed in those isolated from neutrophils responding to the mutant. Together, these data demonstrate that Y. pestis actively inhibits the production of antimicrobial EVs produced by neutrophils, likely contributing to immune evasion.

Legionella species evade degradation and proliferate within alveolar macrophages as an essential step for the manifestation of disease. However, most intracellular bacterial pathogens are restricted in neutrophils, which are the first line of innate immune defense against invading pathogens. Bacterial degradation within neutrophils is mediated by the fusion of microbicidal granules to pathogen-containing phagosomes and the generation of reactive oxygen species (ROS) by the phagocyte NADPH oxidase complex. Here, we show that human neutrophils fail to trigger microbicidal processes and, consequently, fail to restrict L. longbeachae . In addition, neutrophils infected with L. longbeachae fail to undergo a robust pro-inflammatory response, such as degranulation and IL-8 production. Here, we identify three strategies employed by L. longbeachae for evading restriction by neutrophils and inhibiting the neutrophil microbicidal response to other bacteria co-inhabiting in the same cell. First, L. longbeachae excludes the cytosolic and membrane-bound subunits of the phagocyte NADPH oxidase complex from its phagosomal membrane independent of the type 4 secretion system (T4SS). Consequently, infected neutrophils fail to generate robust ROS in response to L. longbeachae . Second, L. longbeachae impedes the fusion of azurophilic granules to its phagosome and the phagosomes of bacteria co-inhabiting the same cell through T4SS-independent mechanisms. Third, L. longbeachae protects phagosomes of co-inhabiting bacteria from degradation by ROS through a trans -acting T4SS-dependent mechanism. Collectively, we conclude that L. longbeachae evades restriction by human neutrophils via T4SS-independent mechanisms and utilizes trans -acting T4SS-dependent mechanisms for inhibition of neutrophil ROS generation throughout the cell cytosol. IMPORTANCE: Legionella longbeachae is commonly found in soil environments where it interacts with a wide variety of protist hosts and microbial competitors. Upon transmission to humans , L. longbeachae invades and replicates within alveolar macrophages, leading to the manifestation of pneumonia. In addition to alveolar macrophages, neutrophils are abundant immune cells acting as the first line of defense against invading pathogens. While most intracellular bacterial species are killed and degraded by neutrophils, we show that L. longbeachae evades degradation. The pathogen impairs the major neutrophils’ microbicidal processes, including the fusion of microbicidal granules to the pathogen-containing vacuole. By inhibiting of assembly of the phagocyte NADPH oxidase complex, the pathogen blocks neutrophils from generating microbicide reactive oxygen species. Overall, L. longbeachae employs unique virulence strategies to evade the major microbicidal processes of neutrophils.

Neutrophils, particularly low-density neutrophils (LDNs), are believed to contribute to acute COVID-19 severity. Here, we showed that neutrophilia can be detected acutely and even months after SARS-CoV-2 infection in patients and mice, while neutrophil depletion reduced disease severity in mice. A key factor in neutrophilia and severe disease in infected mice was traced to the chemokine CXCL12 secreted by bone marrow cells and unexpectedly, endothelial cells. CXCL12 levels were negatively correlated with LDN numbers in longitudinal analyses of patient blood samples. CXCL12 blockade in SARS-CoV-2–infected mice increased blood/lung neutrophil numbers, thereby accelerating disease progression without changing lung virus titers. The exaggerated mortality caused by CXCL12 blockade could be reversed by neutrophil depletion. In addition, blocking interactions between SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2) reduced CXCL12 levels, suggesting a signal transduction from virus-mediated ACE2 ligation to increased CXCL12 secretion. Collectively, these results demonstrate a previously unappreciated role of CXCL12 in diminishing neutrophilia, including low-density neutrophilia, and its deleterious effects in SARS-CoV-2 infections. The results also support the involvement of SARS-CoV-2–endothelial cell interactions in viral pathogenesis.

Yersinia pestis is the etiologic agent of the plague. A hallmark of plague is subversion of the host immune response by disrupting host signaling pathways required for inflammation. This non-inflammatory environment permits bacterial colonization and has been shown to be essential for disease manifestation. Previous work has shown that Y. pestis inhibits phagocytosis and degranulation by neutrophils. Manipulation of these key vesicular trafficking pathways suggests that Y. pestis influences EV secretion, cargo selection, trafficking, and/or maturation. Our goal was to define the EV population produced by neutrophils in response to Y. pestis and determine how these vesicles might influence inflammation. Toward these goals, EVs were isolated from human neutrophils infected with Y. pestis or a mutant lacking bacterial effector proteins known to manipulate host cell signaling. Mass spectrometry data revealed that cargoes packaged in EVs isolated from mutant infected cells were enriched with antimicrobials and cytotoxic proteins, contents which differed from uninfected and Y. pestis infected cells. Further, EVs produced in response to Y. pestis lacked inflammatory properties observed in those isolated from neutrophils responding to the mutant. Together, these data demonstrate that Y. pestis actively inhibits the production of antimicrobial EVs produced by neutrophils, likely contributing to immune evasion.

Summary: Neutrophils are of key importance in periodontal health and disease. In their absence or when they are functionally defective, as occurs in certain congenital disorders, affected individuals develop severe forms of periodontitis in early age. These observations imply that the presence of immune‐competent neutrophils is essential to homeostasis. However, the presence of supernumerary or hyper‐responsive neutrophils, either because of systemic priming or innate immune training, leads to imbalanced host–microbe interactions in the periodontium that culminate in dysbiosis and inflammatory tissue breakdown. These disease‐provoking imbalanced interactions are further exacerbated by periodontal pathogens capable of subverting neutrophil responses to their microbial community's benefit and the host's detriment. This review attempts a synthesis of these findings for an integrated view of the neutrophils' ambivalent role in periodontal disease and, moreover, discusses how some of these concepts underpin the development of novel therapeutic approaches to treat periodontal disease.

Periodontitis is a dysbiotic disease caused by the interplay between the microbial ecosystem present in the disease with the dysregulated host immune response. The disease-associated microbial community is formed by the presence of established oral pathogens like Aggregatibacter actinomycetemcomitans as well as by newly dominant species like Filifactor alocis. These two oral pathogens prevail and grow within the periodontal pocket which highlights their ability to evade the host immune response. This review focuses on the virulence factors and potential pathogenicity of both oral pathogens in periodontitis, accentuating the recent description of F. alocis virulence factors, including the presence of an exotoxin, and comparing them with the defined factors associated with A. actinomycetemcomitans. In the disease setting, possible synergistic and/or mutualistic interactions among both oral pathogens might contribute to disease progression.