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Polyomavirus BKV is highly prevalent among humans. The virus establishes an asymptomatic persistent infection in the urinary system in healthy people, but uncontrolled productive infection of the virus in immunocompromised patients can lead to serious diseases. In spite of its high prevalence, our knowledge regarding key aspects of BKV polyomavirus infection remains incomplete. To determine tissue and cell type tropism of the virus, primary human epithelial cells, endothelial cells and fibroblasts isolated from the respiratory and urinary systems were tested. Results from this study demonstrated that all 9 different types of human cells were infectable by BKV polyomavirus but showed differential cellular responses. In microvascular endothelial cells from the lung and the bladder, BKV persistent infection led to prolonged viral protein expression, low yield of infectious progeny and delayed cell death, in contrast with infection in renal proximal tubular epithelial cells, a widely used cell culture model for studying productive infection of this virus. Transcriptomic profiling revealed the activation of interferon signaling and induction of multiple interferon stimulated genes in infected microvascular endothelial cells. Further investigation demonstrated production of IFNβ and secretion of chemokine CXCL10 by infected endothelial cells. Activation of IRF3 and STAT1 in infected endothelial cells was also confirmed. In contrast, renal proximal tubular epithelial cells failed to mount an interferon response and underwent progressive cell death. These results demonstrated that microvascular endothelial cells are able to activate interferon signaling in response to polyomavirus BKV infection. This raises the possibility that endothelial cells might provide initial immune defense against BKV infection. Our results shed light on the persistence of and immunity against infection by BKV polyomavirus.

Influenza-associated bacterial superinfections in the lung lead to increased morbidity and mortality. Nearly all people have preexisting memory to influenza virus, which can protect against subsequent infection in the lung. This study explored the role B cells play in protection against bacterial (Staphylococcus aureus or Klebsiella pneumoniae) superinfection with previous heterotypic influenza memory. B cell deficiency resulted in an increased inflammatory lung environment and lung tissue injury during superinfection. Loss of B cells increased populations of memory CD8+ T cells in the lung, and these CD8+ T cells were transcriptionally and functionally distinct from those of WT mice. Use of antibody-deficient mouse models showed that this phenotype was specifically due to loss of antibody production from B cells. Passive immunization with influenza antibody serum in B cell-deficient mice rescued the CD8+ T cell phenotype. CD8+ T cell depletion and lethal superinfection challenge experiments showed that the cytotoxic memory CD8+ T cells from B cell-deficient mice protect against superinfection bacterial burden and mortality. These findings provide insight into the importance of B cells for regulating immune responses against infection.

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1 -/- mice with influenza A/PR/8/34 followed by S . aureus USA300. We demonstrated that global IFNLR1 -/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX 3 CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

Viral infections increase host susceptibility to opportunistic pathogens like Aspergillus fumigatus (AF), exacerbating disease severity and prolonging its clinical course. Interleukin‐6 (IL‐6) drives pathological inflammation in viral infections such as COVID‐19, but its role in influenza, particularly with secondary AF infection, remains unclear. Using a mouse model of post‐influenza AF infection, including IL‐6 knockout mice, we found that IL‐6 signaling promotes neutrophilic lung inflammation but is not required for pathogen clearance of either influenza or AF. However, IL‐6 deficiency increases epithelial cell damage, as indicated by elevated RAGE levels in bronchoalveolar lavage fluid. In contrast, lung capillary permeability (measured by IgM levels in BAL) and tissue injury (assessed histologically) remain unaffected in the absence of IL‐6 signaling. These findings reveal a nuanced role for IL‐6 in post‐influenza AF infection, underscoring its contribution to lung inflammation and epithelial integrity.

Influenza infections are often complicated by secondary bacterial infections such as MRSA pneumonia, which increase morbidity and mortality. Viral infections lead to an inflammatory response that includes elevated levels of IL‐6 and interferons. IL‐6 activates the JAK/STAT signaling pathway, amplifying downstream inflammation. Given the clinical efficacy of the JAK inhibitor baricitinib in reducing disease severity in COVID‐19, we evaluated its impact in a murine model of influenza, MRSA, and post‐influenza MRSA pneumonia. Additionally, because IL‐6 inhibitory therapies have improved outcomes during COVID‐19, we evaluated the impact of IL‐6 deletion on post‐influenza MRSA pneumonia. In our studies, baricitinib effectively inhibited the JAK/STAT pathway in the lungs, as demonstrated by decreased interferon‐stimulated genes (ISGs) and STAT3 phosphorylation. Despite this inhibition, baricitinib did not cause a global suppression of cytokines. Notably, baricitinib treatment did not impair either antiviral or antibacterial host immunity, inflammatory cell recruitment, or lung tissue injury. IL‐6 deficiency did not alter weight loss, inflammatory cell recruitment, or bacterial burden during post‐influenza MRSA pneumonia. These findings suggest that both JAK inhibition via baricitinib and IL‐6 deletion do not enhance host defense or limit tissue injury in murine models of influenza and post‐influenza MRSA pneumonia.

The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy. Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.

Influenza-associated bacterial superinfections in the lung lead to increased morbidity and mortality. Nearly all people have preexisting memory to influenza virus, which can protect against subsequent infection in the lung. This study explored the role B cells play in protection against bacterial (Staphylococcus aureus or Klebsiella pneumoniae) superinfection with previous heterotypic influenza memory. B cell deficiency resulted in an increased inflammatory lung environment and lung tissue injury during superinfection. Loss of B cells increased populations of memory [CD8.sup.+] T cells in the lung, and these [CD8.sup.+] T cells were transcriptionally and functionally distinct from those of WT mice. Use of antibody-deficient mouse models showed that this phenotype was specifically due to loss of antibody production from B cells. Passive immunization with influenza antibody serum in B cell-deficient mice rescued the [CD8.sup.+] T cell phenotype. [CD8.sup.+] T cell depletion and lethal superinfection challenge experiments showed that the cytotoxic memory [CD8.sup.+] T cells from B cell-deficient mice protect against superinfection bacterial burden and mortality. These findings provide insight into the importance of B cells for regulating immune responses against infection.

IntroductionInfluenza A is a virus from the Orthomixoviridae family responsible for high lethality rates and morbidity, despite clinically proven vaccination strategies and some anti-viral therapies. The eicosanoid Lipoxin A4 (LXA4) promotes the resolution of inflammation by decreasing cell recruitment and pro-inflammatory cytokines release, but also for inducing activation of apoptosis, efferocytosis, and macrophage reprogramming.ObjectiveHere, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model.MethodMice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 μg/kg/day, i.p.) at day 3 post-infection.ResultsAT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in fpr2/3 −/− animals. In mice treated with LXA4 (50 μg/kg/day, i.p., days 3–6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of T helper 2 cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice.ConclusionTherefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

The question of whether some cases of interstitial cystitis may have an infectious etiology has been debated for some time. Previous studies have looked for the presence of certain specific viruses, but generally did not use the types of sensitive and unbiased approaches that are currently available. As part of the MAPP (Multidisciplinary Approach to the Study of Chronic Pelvic Pain) Research Network, we examined urine specimens from interstitial cystitis patients who provided specimens over time and also reported various symptoms at the time of urine collection. We first performed next-generation sequencing to look for the presence of viruses in urines, and detected two human polyomaviruses that are known to be excreted into urine, BKPyV and JCPyV. We were especially interested in BKPyV because it is a known cause of another bladder disease, hemorrhagic cystitis, in bone marrow transplant recipients. Further analysis of individual samples indicates a trend toward higher excretion of polyomaviruses in patients experiencing increased symptoms.

The necessity of extracellular Ca.sup.2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca.sup.2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca.sup.2+ chelator BAPTA. Here we sought to resolve this controversy. Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca.sup.2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca.sup.2+ chelator, or with BAPTA and saturating CaCl.sub.2 . Together these results indicate that fertilization does not occur in high concentrations of Ca.sup.2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca.sup.2+. Taken together, these data are consistent with requirement of extracellular Ca.sup.2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca.sup.2+ ions to foster fertilization in changing extracellular milieu.