BTK inhibition modulates multiple immune cell populations involved in the pathogenesis of immune mediated nephritis

Highlights

• Mechanisms of nephritis remission and disease flares were explored in nephrotoxic nephritis.

• Remission correlated with a decrease in inflammatory macrophages and T cells.

• An increase in the representation of resident macrophages favored remission.

• Renal flares were associated with increased infiltration of CD45+ myeloid cells.

• Increased expression of IL-6, CD14, and CCL2 were associated with disease flare.

Abstract

Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus. Nephrotoxic serum nephritis (NTN) is an inducible model of LN, which utilizes passive transfer of pre-formed nephrotoxic antibodies to initiate disease. In previous studies, we demonstrated that the Bruton's tyrosine kinase inhibitor, BI-BTK-1, prevents the development of nephritis in NTN when treatment was started prior to nephrotoxic serum transfer, and reverses established proteinuria as well.

We manipulated the initiation and duration of BI-BTK-1 therapy in NTN to study its delayed therapeutic effects when treatment is given later in the disease course, as well as to further understand what effect BI-BTK-1 is having to prevent initiation of nephritis with early treatment. Early treatment and remission induction each correlated with decreased inflammatory macrophages, CD4+ and CD8+ T cells, and decreased B220+ B cells. Additionally, an increased proportion of resident macrophages within the CD45+ population favored a delay of disease onset and remission induction.

We also studied the cellular processes involved in reactivation of nephritis by withdrawing BI-BTK-1 treatment at different time points. Treatment cessation led to either early or later onset of renal flares inversely dependent on the initial duration of BTK inhibition, as assessed by increased proteinuria and BUN levels and worse renal pathology. These flares were associated with an increase in kidney CD45+ infiltrates, including myeloid cell populations. IL-6, CD14, and CCL2 were also increased in mice developing late flares. These analyses point to the role of macrophages as an important contributor to the pathogenesis of immune mediated nephritis, and further support the therapeutic potential of BTK inhibition in this disease and related conditions.

Introduction

Involvement of the kidneys (lupus nephritis, LN) can occur in upwards of 60% of patients with systemic lupus erythematosus (SLE), adding considerable morbidity and mortality to the disease [1]. Current therapies are less than ideal, and the search for more targeted and efficacious treatments is ongoing [2]. One promising therapeutic target is Bruton's tyrosine kinase (BTK). Targeting BTK with a highly selective inhibitor, BI-BTK-1 (or other BTK inhibitors) demonstrates efficacy in multiple murine models of LN, including the nephrotoxic serum nephritis (NTN) model, an inducible model of LN [3,4].

The pathogenesis of LN involves immune complex deposition, infiltration of immune cells into the kidney, and robust expression of inflammatory cytokines. Underlying initiation and progression of disease is the complicated orchestration of many different immune cell types including B cells and macrophages [5]. Further understanding of how these cell types contribute to both disease flare and remission would be very beneficial in the design of targeted new therapies.

BTK significantly affects many different cellular compartments relevant to the pathogenesis of LN. While predominantly known for its role in B cell and macrophage function [[6], [7], [8]], BTK has also recently been shown to be important for certain T cell functions [9], as well as neutrophil recruitment [10], and regulation of dendritic cell maturation [11]. Within the context of SLE, B cells produce the pathogenic autoantibodies associated with the development of disease, and contribute to the inflammatory environment through cytokine release. Macrophages activate when sensing autoantibody deposition in the kidneys through their Fc gamma receptors, and consequently contribute as well to the inflammatory environment and participate in tissue damage. T cells [12,13], neutrophils [14], and dendritic cells [13] have also been implicated in LN, as well as within the NTN model of the disease.

In previous studies using the NTN model, we demonstrated that BI-BTK-1 can not only prevent the development of disease if given prior to the nephrotoxic serum transfer, but that it can also reverse already established disease [3]. In light of this established efficacy of BI-BTK-1 treatment, we sought to study the pathophysiology of the induction of remission when treatment is given later in the disease course, as well as to further understand what effect BI-BTK-1 is having to prevent initiation of nephritis. We also wanted to study the cellular processes that lead to clinical expression of a disease flare. This information could provide important and clinically relevant insights into the use of BTK inhibitors at different stages of LN, as well as suggest other potential therapeutic targets for future development.

We designed two studies where BTK inhibition was utilized to study the pathogenesis of both LN remission and flares. In the first experiment, we staggered the beginning of treatment, and then monitored nephritis development over time. Using staggered sacrifices to allow detailed assessment of different time points in the disease course, we sought to illuminate how disease is prevented with early treatment and how remission is induced with late treatment. In the second experiment, we utilized the withdrawal of treatment either early or late in disease to study the kinetics of flare induction.