Sheppard, Dean

AbstractBackgroundAPOE ε4 is the strongest genetic risk factor for late‐onset Alzheimer’s disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD).MethodGeneration of CX3CR1‐CREERT2 mice crossed to APOE‐KI(E3 and E4)fl/fl: APP/PS1 and APOE‐KI(E3 and E4)fl/fl: P301S mice. Microglia and astrocytes were isolated and sequenced using either bulk RNA‐seq (MGnD‐Paradigm) or single‐cell RNA‐seq. Crosstalk between cells was determined using the NichenetR database and validated using 1) recombinant Lgals3 intracranial injection into APP/PS1 mice and 2) adoptive transfer of microglia, from APOE3‐KI, APOE4‐KI, and APOE4‐cKO mice challenged with labeled apoptotic neurons, into WT mice and isolation of astrocytes. Validation in the brain of AD donors carrying the APOE e3/3 and e3/4 alleles.ResultHere we show, in mice and in humans, a negative role of microglial APOE4 in the induction of MGnD response to neurodegeneration. Deletion of microglial APOE4 restores MGnD phenotype, associated with neuroprotection in P301S tau transgenic mice, and decreases pathology in APP/PS1 mice. MGnD‐astrocyte crosstalk associated with b‐amyloid (Ab) plaque encapsulation and clearance is mediated via Lgals3 signaling following microglial APOE4 deletion. In the brain of AD donors carrying the APOE ε4 allele, we found a sex‐dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, as compared to APOE ε3/3 carriers. Mechanistically, APOE4‐mediated induction of ITGB8‐TGFb signaling impairs MGnD response via upregulation of TGFb‐mediated microglial homeostatic checkpoints, including INPP5D. Microglial deletion of Inpp5d restores MGnD‐astrocyte crosstalk and facilitates plaque clearance in APP/PS1 mice. Genetic and pharmacological blocking of ITGB8‐TGFb signaling enhanced MGnD response associated with increased plaque clearance in AD mice.ConclusionWe identify the microglial APOE4‐ITGB8‐TGFb pathway as a negative regulator of microglia‐astrocyte response to AD pathology, and restoring MGnD phenotype via blocking ITGB8‐TGFb signaling may provide new molecular targets to modulate and restore functional microglia in AD.

Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.

Abstract Rationale Transforming growth factor (TGF)-β has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin αvβ6, a key activator of TGF-β in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-β at sites of αvβ6 up-regulation without affecting other homeostatic roles of TGF-β. Objectives To analyze the expression of αvβ6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of αvβ6-mediated TGF-β activation in murine bleomycin–induced pulmonary fibrosis. Methods Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for αvβ6 expression. A range of concentrations of a monoclonal antibody that blocks αvβ6-mediated TGF-β activation was evaluated in murine bleomycin–induced lung fibrosis. Measurements and Main Results αvβ6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, αvβ6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-β inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers. Conclusions Partial inhibition of TGF-β using αvβ6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of αvβ6, an activator of the fibrogenic cytokine, TGF-β, in human pulmonary fibrosis suggests that αvβ6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.