Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

Practical Mouse Model to Investigate Therapeutics for Staphylococcusaureus Contaminated Surgical Mesh Implants.

INTRODUCTION: The use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus-infected surgical mesh and provide results from antibiotic and immunotherapeutic testing. MATERIALS AND METHODS: Implantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 "don't eat me" molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points. RESULTS: A single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection. CONCLUSIONS: This fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.

Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques.

Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques.

Severe coronavirus disease 2019 (COVID-19) has been associated with T cell lymphopenia, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we studied rhesus macaques that were depleted of either CD4+, CD8+, or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to that in controls. The T cell-depleted groups developed virus-neutralizing antibody responses and class switched to IgG. When reinfected 6 weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads, and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ nor CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory, or protection from a second infection. IMPORTANCE Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it. We studied rhesus macaques, which develop only mild COVID-19, similar to most humans. Experimental depletion of T cells slightly prolonged their clearance of virus, but there was no increase in disease severity. Furthermore, they were able to develop protection from a second infection and produced antibodies capable of neutralizing the virus. They also developed immunological memory, which allows a much stronger and more rapid response upon a second infection. These results suggest that T cells are not critical for recovery from acute SARS-CoV-2 infections in this model and point toward B cell responses and antibodies as the essential mediators of protection from re-exposure.

The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction.

Staphylococcus aureus (S. aureus) causes a range of diseases ranging from superficial skin and soft-tissue infections to invasive and life-threatening conditions (Klevens et al., 2007; Kobayashi et al., 2015). S. aureus utilizes the Sae sensory system to adapt to neutrophil challenge. Although the roles of the SaeR response regulator and its cognate sensor kinase SaeS have been demonstrated to be critical for surviving neutrophil interaction and for causing infection, the roles for the accessory proteins SaeP and SaeQ remain incompletely defined. To characterize the functional role of these proteins during innate immune interaction, we generated isogenic deletion mutants lacking these accessory genes in USA300 (USA300ΔsaeP and USA300ΔsaeQ). S. aureus survival was increased following phagocytosis of USA300ΔsaeP compared to USA300 by neutrophils. Additionally, secreted extracellular proteins produced by USA300ΔsaeP cells caused significantly more plasma membrane damage to human neutrophils than extracellular proteins produced by USA300 cells. Deletion of saeQ resulted in a similar phenotype, but effects did not reach significance during neutrophil interaction. The enhanced cytotoxicity of USA300ΔsaeP cells toward human neutrophils correlated with an increased expression of bi-component leukocidins known to target these immune cells. A saeP and saeQ double mutant (USA300ΔsaePQ) showed a significant increase in survival following neutrophil phagocytosis that was comparable to the USA300ΔsaeP single mutant and increased the virulence of USA300 during murine bacteremia. These data provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeP and saeQ together impact pathogenesis in vivo.

Aspartic Acid Residue 51 of SaeR Is Essential for Staphylococcus aureus Virulence.

Staphylococcus aureus is a common Gram-positive bacteria that is a major cause of human morbidity and mortality. The SaeR/S two-component sensory system of S. aureus is important for virulence gene transcription and pathogenesis. However, the influence of SaeR phosphorylation on virulence gene transcription is not clear. To determine the importance of potential SaeR phosphorylation sites for S. aureus virulence, we generated genomic alanine substitutions at conserved aspartic acid residues in the receiver domain of the SaeR response regulator in clinically significant S. aureus pulsed-field gel electrophoresis (PFGE) type USA300. Transcriptional analysis demonstrated a dramatic reduction in the transcript abundance of various toxins, adhesins, and immunomodulatory proteins for SaeR with an aspartic acid to alanine substitution at residue 51. These findings corresponded to a significant decrease in cytotoxicity against human erythrocytes and polymorphonuclear leukocytes, the ability to block human myeloperoxidase activity, and pathogenesis during murine soft-tissue infection. Analysis of SaeR sequences from over 8,000 draft S. aureus genomes revealed that aspartic acid residue 51 is 100% conserved. Collectively, these results demonstrate that aspartic acid residue 51 of SaeR is essential for S. aureus virulence and underscore a conserved target for novel antimicrobial strategies that treat infection caused by this pathogen.

Differential regulation of CD103 (αE integrin) expression in human dendritic cells by retinoic acid and Toll-like receptor ligands.

CD103 (αE integrin) is an important dendritic cell (DC) marker that characterizes functionally distinct DC subsets in mice and humans. However, the mechanism by which CD103 expression is regulated in human DCs and the role of CD103 for DC function are not very well understood. Here, we show that retinoic acid (RA) treatment of human monocyte-derived DCs (MoDCs) increased the ability of the DCs to synthesize RA and induced MoDC expression of CD103 and ß7 at the mRNA and protein level. In contrast, RA was unable to induce the expression of CD103 in primary human DCs isolated from the gastric mucosa. Inhibition of TGF-ß signaling in MoDCs down-regulated RA-induced CD103 expression, indicating that TGF-ß-dependent pathways contribute to the induction of CD103. Conversely, when RA-treated MoDCs were stimulated with live Helicobacter pylori, commensal bacteria, LPS, or a TLR2 agonist, the RA-induced up-regulation of CD103 and ß7 integrin expression was completely abrogated. To determine whether CD103 expression impacts DC priming of CD4+ T cells, we next investigated the ability of CD103+ and CD103─ DCs to induce mucosal homing and T cell proliferation. Surprisingly, RA treatment of DCs enhanced both α4ß7 expression and proliferation in cocultured T cells, but no difference was seen between RA-treated CD103+ and CD103─ DCs. In summary, our data demonstrate that RA, bacterial products, and the tissue environment all contribute to the regulation of CD103 on human DCs and that DC induction of mucosal homing in T cells is RA dependent but not CD103 dependent.