No abstract

Spatial information of cells in their tissue microenvironment is necessary to understand the complexity of pathophysiological processes. Volumetric imaging of cleared organs provides this information; however, current protocols are often elaborate, expensive, and organ specific. We developed a simplified, cost-effective, non-hazardous approach for efficient tissue clearing and multi-organ volumetric imaging (EMOVI). EMOVI enabled multiplexed antibody-based immunolabeling, provided adequate tissue transparency, maintained cellular morphology and preserved fluorochromes. Exemplarily, EMOVI allowed the detection and quantification of scarce cell populations during pneumonitis. EMOVI also permitted histo-cytometric analysis of MHC-II expressing cells, revealing distinct populations surrounding or infiltrating glomeruli of nephritic kidneys. Using EMOVI, we found widefield microscopy with real-time computational clearing as a valuable option for rapid image acquisition and detection of rare cellular events in cleared organs. EMOVI has the potential to make tissue clearing and volumetric imaging of immune cells applicable for a broad audience by facilitating flexibility in organ, fluorochrome and microscopy usage.

Dysregulated B cell responses have been described in inflammatory-bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely understood. We here described Wiskott-Aldrich Syndrome interacting protein deficient (Wipf1 -/-) mice as novel mouse model of spontaneous, chronic colitis modelling human IBD. Concomitant with aberrant IgG production in colonic tissue of Wipf1 -/mice, we identified systemic, hypo-sialylated IgG as drivers of IL-1 production in monocytes. Pathological antibody production was promoted by the hyper-reactivity of Wipf1 -/-B cells in response to LPS stimulation, resulting in efficient activation of the MAPK/Erk and mTOR/Akt/4E-BP1 pathways and heightened metabolic activity. In addition to abundant inflammatory IgG, we found that B cells directly promoted the production of pro-inflammatory cytokines by intestinal CD4 + T cells. B/T co-culture assays defined the co-stimulatory molecule CD86 as driver of IFN- and GM-CSF production by CD4 + T cells. CD86 expression was further enhanced by the presence of sCD40L, which was elevated in sera of Wipf1 -/mice. Similarly, colonic B cells of IBD patients expressed increased mRNA levels of CD86 correlating with enhanced levels of systemic sCD40L. Together, B cellmediated pro-inflammatory cytokine secretion and B cell-derived inflammatory antibody production contributed to exacerbated pathogenesis during intestinal inflammation..