P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

SCGB1D2 inhibits growth of Borrelia burgdorferi and affects susceptibility to Lyme disease.

Lyme disease is a tick-borne disease caused by bacteria of the genus Borrelia. The host factors that modulate susceptibility for Lyme disease have remained mostly unknown. Using epidemiological and genetic data from FinnGen and Estonian Biobank, we identify two previously known variants and an unknown common missense variant at the gene encoding for Secretoglobin family 1D member 2 (SCGB1D2) protein that increases the susceptibility for Lyme disease. Using live Borrelia burgdorferi (Bb) we find that recombinant reference SCGB1D2 protein inhibits the growth of Bb in vitro more efficiently than the recombinant protein with SCGB1D2 P53L deleterious missense variant. Finally, using an in vivo murine infection model we show that recombinant SCGB1D2 prevents infection by Borrelia in vivo. Together, these data suggest that SCGB1D2 is a host defense factor present in the skin, sweat, and other secretions which protects against Bb infection and opens an exciting therapeutic avenue for Lyme disease.

Distinct, direct and climate-mediated environmental controls on global particulate and mineral-associated organic carbon storage.

Identifying controls on soil organic carbon (SOC) storage, and where SOC is most vulnerable to loss, are essential to managing soils for both climate change mitigation and global food security. However, we currently lack a comprehensive understanding of the global drivers of SOC storage, especially with regards to particulate (POC) and mineral-associated organic carbon (MAOC). To better understand hierarchical controls on POC and MAOC, we applied path analyses to SOC fractions, climate (i.e., mean annual temperature [MAT] and mean annual precipitation minus potential evapotranspiration [MAP-PET]), carbon (C) input (i.e., net primary production [NPP]), and soil property data synthesized from 72 published studies, along with data we generated from the National Ecological Observatory Network soil pits (n = 901 total observations). To assess the utility of investigating POC and MAOC separately in understanding SOC storage controls, we then compared these results with another path analysis predicting bulk SOC storage. We found that POC storage is negatively related to MAT and soil pH, while MAOC storage is positively related to NPP and MAP-PET, but negatively related to soil % sand. Our path analysis predicting bulk SOC revealed similar trends but explained less variation in C storage than our POC and MAOC analyses. Given that temperature and pH impose constraints on microbial decomposition, this indicates that POC is primarily controlled by SOC loss processes. In contrast, strong relationships with variables related to plant productivity constraints, moisture, and mineral surface availability for sorption indicate that MAOC is primarily controlled by climate-driven variations in C inputs to the soil, as well as C stabilization mechanisms. Altogether, these results demonstrate that global POC and MAOC storage are controlled by separate environmental variables, further justifying the need to quantify and model these C fractions separately to assess and forecast the responses of SOC storage to global change.

SIRPα controls CD47-dependent platelet clearance in mice and humans.

Over the last decade, more data has revealed that increased surface expression of the "don't eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRPα can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRPα signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRPα axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRPα axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy.

Ammonia-oxidizing archaea and bacteria differentially contribute to ammonia oxidation in soil under precipitation gradients and land legacy.

Background: Global change has accelerated the nitrogen cycle. Soil nitrogen stock degradation by microbes leads to the release of various gases, including nitrous oxide (N2O), a potent greenhouse gas. Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) participate in the soil nitrogen cycle, producing N2O. There are outstanding questions regarding the impact of environmental processes such as precipitation and land use legacy on AOA and AOB structurally, compositionally, and functionally. To answer these questions, we analyzed field soil cores and soil monoliths under varying precipitation profiles and land legacies. Results: We resolved 28 AOA and AOB metagenome assembled genomes (MAGs) and found that they were significantly higher in drier environments and differentially abundant in different land use legacies. We further dissected AOA and AOB functional potentials to understand their contribution to nitrogen transformation capabilities. We identified the involvement of stress response genes, differential metabolic functional potentials, and subtle population dynamics under different environmental parameters for AOA and AOB. We observed that AOA MAGs lacked a canonical membrane-bound electron transport chain and F-type ATPase but possessed A/A-type ATPase, while AOB MAGs had a complete complex III module and F-type ATPase, suggesting differential survival strategies of AOA and AOB. Conclusions: The outcomes from this study will enable us to comprehend how drought-like environments and land use legacies could impact AOA- and AOB-driven nitrogen transformations in soil.

Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody.

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

CD47 Blockade Leads to Chemokine-Dependent Monocyte Infiltration and Loss of B Cells from the Splenic Marginal Zone.

CD47 is an important innate immune checkpoint through its interaction with its inhibitory receptor on macrophages, signal-regulatory protein α (SIRPα). Therapeutic blockade of CD47-SIRPα interactions is a promising immuno-oncology treatment that promotes clearance of cancer cells. However, CD47-SIRPα interactions also maintain homeostatic lymphocyte levels. In this study, we report that the mouse splenic marginal zone B cell population is dependent on intact CD47-SIRPα interactions and blockade of CD47 leads to the loss of these cells. This depletion is accompanied by elevated levels of monocyte-recruiting chemokines CCL2 and CCL7 and infiltration of CCR2+Ly6Chi monocytes into the mouse spleen. In the absence of CCR2 signaling, there is no infiltration and reduced marginal zone B cell depletion. These data suggest that CD47 blockade leads to clearance of splenic marginal zone B cells.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Recurrent fires do not affect the abundance of soil fungi in a frequently burned pine savanna.

While the negative effects of infrequent, high-intensity fire on soil fungal abundance are well-understood, it remains unclear how the short-term history of frequent, low-intensity fire in fire-dependent ecosystems impacts abundance, and whether this history governs any abundance declines. We used prescribed fire to experimentally alter the short-term fire history of patches within a fire-frequented old-growth pine savanna over a 3 y period. We then quantified fungal abundance before and after the final fire using phospholipid fatty acid (PLFA) assays and Droplet Digital™ PCR (ddPCR). Short-term fire history largely did not affect total fungal abundance nor pre- to post-fire abundance shifts. While producing similar conclusions, PLFA and ddPCR data were not correlated. In addition to piloting a new method to quantify soil fungal abundance, our findings indicate that, within fire-frequented pine savannas, recurrent fires do not consistently decrease total fungal abundance, and abundance changes are not contingent upon short-term fire history. This suggests that many fungi in fire-dependent ecosystems are fire-tolerant.