Multispectral imaging (MSI) is increasingly finding application in the study and characterization of biological specimens. However, the methods typically used come with challenges on both the acquisition and the analysis front. MSI can be slow and photon-inefficient, leading to long imaging times and possible phototoxicity and photobleaching. The resulting datasets can be large and complex, prompting the development of a number of mathematical approaches for segmentation and signal unmixing. We show that under certain circumstances, just three spectral channels provided by standard color cameras, coupled with multispectral analysis tools, including a more recent spectral phasor approach, can efficiently provide useful insights. These findings are supported with a mathematical model relating spectral bandwidth and spectral channel number to achievable spectral accuracy. The utility of 3-band RGB and MSI analysis tools are demonstrated on images acquired using brightfield and fluorescence techniques, as well as a novel microscopy approach employing UV-surface excitation. Supervised linear unmixing, automated non-negative matrix factorization and phasor analysis tools all provide useful results, with phasors generating particularly helpful spectral display plots for sample exploration.

Patients with autoimmune diseases are more susceptible to foodborne infections, which can be exacerbated by immunosuppressive therapy. Tofacitinib, a JAK/STAT pathway inhibitor, was recently approved for the treatment of ulcerative colitis, yet its effects on the pathogenesis of intestinal infections remain unclear. Here, we examined the impact of oral tofacitinib treatment in a mouse model of Campylobacter jejuni (C. jejuni) infection. Our results show that early tofacitinib administration attenuates intestinal pathology without affecting bacterial colonization. Specifically, tofacitinib suppressed CXCL1, CXCL2, CCL2 chemokine expression by intestinal epithelial cells, limiting recruitment of monocytes and neutrophils to the gut. In addition, JAK/STAT inhibition reduced IFNγ-producing innate lymphoid cells (ILCs) and T cells in the gut. Furthermore, tofacitinib suppressed IFNγ production and ameliorated intestinal disease in humanized mice. Cell-fate mapping revealed that tofacitinib predominantly inhibited IFNγ production by NK1.1^- ILCs derived from NKp46^- progenitors and reduced NK1.1^- ILC proliferation without affecting ILC3 to ILC1 plasticity. Notably, tofacitinib ameliorated intestinal disease even in the absence of T cells. These findings suggest that tofacitinib alleviates C. jejuni-induced colitis by reducing proinflammatory cytokine production by monocytes/macrophages/epithelial cells and suppressing IFNγ secretion by ILCs and T cells, while preserving antibacterial defenses.

MUSE rapidly creates high-resolution, histology images from thick specimens using off-axis 280-nm UV excitation and exogenous dyes. These emit in their usual visible range; the resulting images can be captured using ordinary lenses and color cameras.

Streptococcus pneumoniae (Spn) is a common bacterial co-infection in patients with respiratory viral infections, including influenza and SARS-CoV-2, and is associated with increased morbidity and mortality. However, the mechanisms underlying the severe disease caused by SARS-CoV-2 and Spn co-infection remain poorly understood. Here, we show that Spn infection 3 days after infection with mouse-adapted SARS-CoV-2 MA10 markedly increased morbidity and mortality in wild-type mice. In contrast, SARS-CoV-2 infection 3 days after Spn infection did not increase mortality, demonstrating that disease severity depends on the sequence of infection. Increased susceptibility to Spn following SARS-CoV-2 infection was associated with enhanced viral burden, exacerbated lung pathology, pronounced neutrophil accumulation, and increased expression of IFN-β and the neutrophil-recruiting chemokine CXCL2. To determine whether pneumolysin (PLY), a major Spn virulence factor, contributes to superinfection-induced pathology, we infected mice with a PLY-deficient Spn strain. Unexpectedly, PLY deficiency further increased mortality and lung pathology without altering bacterial or viral titers. Immunofluorescence analysis revealed increased accumulation of neutrophils and macrophages in the lungs of mice co-infected with PLY-deficient Spn. These results indicate that Spn superinfection exacerbates SARS-CoV-2-induced lung disease, whereas PLY unexpectedly attenuates disease severity. We propose that PLY limits immunopathology during SARS-CoV-2–Spn superinfection by restraining macrophage- and neutrophil-mediated inflammation.