A specific low-density neutrophil population correlates with hypercoagulation and disease severity in hospitalized COVID-19 patients.

SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19-associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.

Human Neutrophil Granule Exocytosis in Response to Mycobacterium smegmatis.

Mycobacterium smegmatis rarely causes disease in the immunocompetent, but reported cases of soft tissue infection describe abscess formation requiring surgical debridement for resolution. Neutrophils are the first innate immune cells to accumulate at sites of bacterial infection, where reactive oxygen species and proteolytic enzymes are used to kill microbial invaders. As these phagocytic cells play central roles in protection from most bacteria, we assessed human neutrophil phagocytosis and granule exocytosis in response to serum opsonized or non-opsonized M. smegmatis mc2. Although phagocytosis was enhanced by serum opsonization, M. smegmatis did not induce exocytosis of secretory vesicles or azurophilic granules at any time point tested, with or without serum opsonization. At early time points, opsonized M. smegmatis induced significant gelatinase granule exocytosis compared to non-opsonized bacteria. Differences in granule release between opsonized and non-opsonized M. smegmatis decreased in magnitude over the time course examined, with bacteria also evoking specific granule exocytosis by six hours after addition to cultured primary single-donor human neutrophils. Supernatants from neutrophils challenged with opsonized M. smegmatis were able to digest gelatin, suggesting that complement and gelatinase granule exocytosis can contribute to neutrophil-mediated tissue damage seen in these rare soft tissue infections.

Putative Periodontal Pathogens, Filifactor Alocis and Peptoanaerobacter Stomatis, Induce Differential Cytokine and Chemokine Production by Human Neutrophils.

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate host response in the gingival tissue, and the contribution of neutrophil-derived cytokines and chemokines plays a central role in disease progression. The pattern of cytokines and chemokines released by human neutrophils upon stimulation with newly appreciated periodontal bacteria compared to the keystone oral pathogen Porphyromonas gingivalis was investigated. Our results showed that both F. alocis and P. stomatis triggered TLR2/6 activation. F. alocis induced significant changes in gene expression of cytokines and chemokines in human neutrophils compared to unstimulated cells. However, except for IL-1ra, neutrophils released lower levels of cytokines and chemokines in response to F. alocis compared to P. stomatis. Furthermore, bacteria-free conditioned supernatant collected from neutrophils challenged with P. stomatis, but not from P. gingivalis or F. alocis, was chemotactic towards both neutrophils and monocytes. Elucidating stimuli-specific modulation of human neutrophil effector functions in the context of dysbiotic microbial community constituents provides valuable information for understanding the pathogenesis of periodontal diseases.

Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA.

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism . NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

Filifactor alocis modulates human neutrophil antimicrobial functional responses.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F. alocis. Both non-opsonised and serum-opsonised F. alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F. alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F. alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F. alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F. alocis with the same efficiency, heat-killed F. alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F. alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F. alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Do leprosy and tuberculosis generate a systemic inflammatory shift? Setting the ground for a new dialogue between experimental immunology and bioarchaeology.

It is possible that during long lasting chronic infections such as tuberculosis (TB) and leprosy individuals who generate a stronger immune response will produce a chronic shift in the systemic levels of inflammatory proteins. Consequently, the systemic immunological shift could affect inflammatory responses against other persistent pathogens such as Porphyromonas gingivalis associated with periodontal disease (PD). OBJECTIVE: To determine if in vitro exposure to Mycobacterium tuberculosis or M. leprae lysates impacts subsequent immune responses to P. gingivalis; and to propose a new dialogue between experimental immunology and paleopathology. MATERIAL AND METHODS: We sequentially (2 days protocol) exposed peripheral blood mononuclear cells (PBMCs) from healthy donors to bacterial lysates either from M. tuberculosis, or M. leprae, or P. gingivalis. After collecting all supernatants, we measured the expression of immune proteins TNFα and IFNγ using an enzyme-linked immunosorbent assay. RESULTS: Early exposure (day 1) of PBMCs to M. leprae or M. tuberculosis lysates induces an inflammatory shift detected by the increase of TNFα and IFNγ when the same cells are subsequently (day 2) exposed to oral pathogen P. gingivalis. DISCUSSION: By extrapolating these results, we suggest that chronic infections, such as TB and leprosy, could generate a systemic immunological shift that can affect other inflammatory processes such the one present in PD. We propose that the presence and severity of PD should be explored as a proxy for inflammatory status or competence when reconstructing the health profile in past populations.