Biology subjects

Baker, P. J.

Publications and source records attributed to Baker, P. J..

8 recordsLinked to original sources

bioRxiv2025

Protein kinase F regulates the virulence of Mycobacterium tuberculosis

The serine/threonine protein kinase F (PknF) of Mycobacterium tuberculosis (Mtb) has poorly defined targets and functions but is involved in limiting NLRP3 inflammasome activation in murine macrophages and dendritic cells in vitro. The importance of PknF for the virulence of Mtb in vivo is not known. Here, we demonstrate that the Mtb CDC1551 deletion mutant of pknF ({Delta}pknF) expresses significantly increased levels of the lipid pthiocerol dimycoserosate (PDIM), a polyketide lipid with pro-virulence properties. The {Delta}pknF mutant strain, when compared to the Mtb and complemented strains, had a 100-fold increase in growth at day 28 and about a 10-fold increase in growth at days 90-98 in the lungs of mice. The increase in pulmonary bacterial loads after infection with {Delta}pknF strain was conserved even in Nlrp3-deficient mice, arguing that PknF modulates Mtb virulence independently of NLRP3-inflammasome activation in mice. Staining of lung sections revealed increased inflammation in the lungs of {Delta}pknF strain-infected mice when compared to Mtb and the complemented mutant strain. Highly susceptible B6.Sst1S mice displayed decreased host resistance with significantly decreased survival when infected with the {Delta}pknF strain compared to the complemented strain or Mtb. In conclusion, our data suggest that expression of PknF, as a modulator of multiple downstream effector proteins, restricts the virulence of Mtb in the lungs of mice through an NLRP3 inflammasome-independent mechanism but potentially via suppressing expression of the virulence lipid, PDIM. Author summaryThe human pathogen Mycobacterium tuberculosis (Mtb) encodes 11 serine/threonine protein kinases (Pkn A-I,K and L). In vitro, PknF inhibits activation of innate immune responses in macrophages and dendritic cells. The importance of that protein for the virulence of the bacteria in the context of a live animal infection is unknown. We used a hypersusceptible mouse strain to analyze the impact of deleting the pknF gene on the virulence of the bacteria by monitoring the survival of the mice. We show that the {Delta}pknF deletion mutant kills mice faster than wild-type bacteria or {Delta}pknF mutant bacteria expressing a wild-type copy of the pknF gene ({Delta}pknF-C). Next, we analyzed the growth of the different bacterial strains in the infected mice and demonstrated that at day 28 and day 90-98 timepoints the {Delta}pknF mutant bacterial strains showed a significant increase in bacterial burden in the lungs, bronchoalveolar lavage fluid and the spleen. An analysis of the total lipids of the bacterial stocks showed that the {Delta}pknF Mtb bacteria increased expression of the virulence lipid, pthiocerol dimycoserosate (PDIM), suggesting a negative regulation of this lipid by PknF likely affecting bacterial virulence in the lungs of mice.

microbiology↗

bioRxiv2024

The inflammatory microenvironment of the lung at the time of infection governs innate control of SARS-CoV-2 replication

SARS-CoV-2 infection leads to vastly divergent clinical outcomes ranging from asymptomatic infection to fatal disease. Co-morbidities, sex, age, host genetics and vaccine status are known to affect disease severity. Yet, how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure impacts the control of viral replication remains poorly understood. We demonstrate here that immune events in the mouse lung closely preceding SARS-CoV-2 infection significantly impact viral control and we identify key innate immune pathways required to limit viral replication. A diverse set of pulmonary inflammatory stimuli, including resolved antecedent respiratory infections with S. aureus or influenza, ongoing pulmonary M. tuberculosis infection, ovalbumin/alum-induced asthma or airway administration of defined TLR ligands and recombinant cytokines, all establish an antiviral state in the lung that restricts SARS-CoV-2 replication upon infection. In addition to antiviral type I interferons, the broadly inducible inflammatory cytokines TNF and IL-1 precondition the lung for enhanced viral control. Collectively, our work shows that SARS-CoV-2 may benefit from an immunologically quiescent lung microenvironment and suggests that heterogeneity in pulmonary inflammation that precedes or accompanies SARS-CoV-2 exposure may be a significant factor contributing to the population-wide variability in COVID-19 disease outcomes.

immunology↗

bioRxiv2023

Pre-existing interferon gamma conditions the lung to mediate early control of SARS-CoV-2

Interferons (IFNs) are critical for anti-viral host defence. Type-1 and type-3 IFNs are typically associated with early control of viral replication and promotion of inflammatory immune responses; however, less is known about the role of IFN{gamma} in anti-viral immunity, particularly in the context of SARS-CoV-2. We have previously observed that lung infection with attenuated bacteria Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 (SCV2) infection and disease in two mouse models. Assessment of the pulmonary cytokine milieu revealed that iv BCG induces a robust IFN{gamma} response and low levels of IFN{beta}. Here we examined the role of ongoing IFN{gamma} responses due to pre-established bacterial infection on SCV2 disease outcomes in two murine models. We report that IFN{gamma} is required for iv BCG induced reduction in pulmonary viral loads and that this outcome is dependent on IFN{gamma} receptor expression by non-hematopoietic cells. Further analysis revealed that BCG infection promotes the upregulation of interferon-stimulated genes (ISGs) with reported anti-viral activity by pneumocytes and bronchial epithelial cells in an IFN{gamma}-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirmed the importance of IFN{gamma} in these anti-viral effects by demonstrating that the recombinant cytokine itself provides strong protection against SCV2 challenge when administered intranasally. Together, our data show that a pre-established IFN{gamma} response within the lung is protective against SCV2 infection, suggesting that concurrent or recent infections that drive IFN{gamma} may limit the pathogenesis of SCV2 and supporting possible prophylactic uses of IFN{gamma} in COVID-19 management.

immunology↗

bioRxiv2022

Evaluation of endogenous and therapeutic 25-hydroxycholesterols in murine models of pulmonary SARS-CoV-2 infection

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h-/- and Gpr183-/- mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.

immunology↗

bioRxiv2022

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection

Influx of eosinophils into the lungs is typically associated with type-II responses during allergy, fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type-I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice where they contribute to host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb-exposure. In mice this influx was CCR3 independent and instead required cell-intrinsic expression of the oxysterol-receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interacted directly with bacilli-laden alveolar macrophages, which upregulated the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment was impaired in Ch25h deficient mice. Our findings show that eosinophils are among the first cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a novel role for GPR183 in the migration of eosinophils into lung tissue. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/480919v1_ufig1.gif" ALT="Figure 1"> View larger version (105K): org.highwire.dtl.DTLVardef@e8cd29org.highwire.dtl.DTLVardef@1f5769borg.highwire.dtl.DTLVardef@3dce9borg.highwire.dtl.DTLVardef@1c75587_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSIn mice and macaques eosinophils accumulate early in Mtb-infected lungs preceding neutrophils Eosinophils interact with Mtb-infected cells in the alveoli in mice Early pulmonary eosinophil migration occurs independently of CCR3 in mice Early lung migration in mice requires Ch25h and eosinophil-intrinsic GPR183 expression

immunology↗

bioRxiv2021

Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge

Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guerin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.

immunology↗

bioRxiv2021

Eosinophils are an integral component of the pulmonary granulocyte response in Tuberculosis and promote host resistance in mice

Host resistance to Mycobacterium tuberculosis infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. Influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, employing complementary genetic models of eosinophil deficiency, we demonstrate that, in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

immunology↗

bioRxiv2020

PD-1 blockade exacerbates Mycobacterium tuberculosis infection in rhesus macaques

Boosting immune cell function by targeting the co-inhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here we examine the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. Animals treated with PD-1 mAb developed worse disease and higher granuloma bacterial loads compared to isotype control treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in PD-1 treated macaques were not increased in number or function in granulomas, upregulated high levels of CTLA-4 and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of PD-1 treated animals, multiple pro-inflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Lastly, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota prior to infection in individual macaques. Therefore, PD-1-mediated co-inhibition is required for control of Mtb infection in macaques, perhaps due to its role in dampening detrimental inflammation as well as allowing for normal CD4 T cell responses.

immunology↗