OTULIN haploinsufficiency predisposes to environmentally directed inflammation

Abstract

Recently, OTULIN haploinsufficiency was linked to enhanced susceptibility to Staphylococcus aureus infections accompanied by local necrosis and systemic inflammation. The pathogenesis observed in haploinsufficient patients differs from the hyperinflammation seen in classical OTULIN-related autoinflammatory syndrome (ORAS) patients and is characterized by increased susceptibility of dermal fibroblasts to S. aureus alpha toxin-inflicted cytotoxic damage. Immunological abnormalities were not observed in OTULIN haploinsufficient patients, suggesting a non-hematopoietic basis. In this research report, we investigated an Otulin^+/- mouse model after in vivo provocation with lipopolysaccharide (LPS) to explore the potential role of hematopoietic-driven inflammation in OTULIN haploinsufficiency. We observed a hyperinflammatory signature in LPS-provoked Otulin^+/- mice, which was driven by CD64⁺ monocytes and macrophages. Bone marrow-derived macrophages (BMDMs) of Otulin^+/- mice demonstrated higher proinflammatory cytokine secretion after in vitro stimulation with LPS or polyinosinic:polycytidylic acid (Poly(I:C)). Our experiments in full and mixed bone marrow chimeric mice suggest that, in contrast to humans, the observed inflammation was mainly driven by the hematopoietic compartment with cell-extrinsic effects likely contributing to inflammatory outcomes. Using an OTULIN haploinsufficient mouse model, we validated the role of OTULIN in the regulation of environmentally directed inflammation.

Keywords: OTULIN; OTULIN deficiency; OTULIN-related autoinflammatory syndrome; inborn errors in immunity; inflammation.

Figure 1

Otulin^+/− mice have a myeloid-driven hyperinflammatory phenotype after in vivo LPS stimulation. (A) Experimental setup. Wt, wild type; litt, littermate. (B) Serum cytokine levels, n = 2 independent experiments, n = 13–14 mice in each group. (C) Normalized MFI of TNF-α and pro-IL-1β in TNF-α+ or pro-IL-1β+ CD64⁺ monocytes and macrophages to the mean of unstimulated sham-treated Wt litt control mice, n = 2 independent experiments, n = 13–14 mice in each group. (D) Cytokine analysis of supernatant of BMDMs stimulated with LPS 5 ng/mL or Poly(I:C) 1.25 μg/mL for 24 hours, n = 2 independent experiments; each dot represents the mean of a technical duplicate, n = 6 biological replicates. IL-1β was undetectable in the Poly(I:C) condition; hence, data are not shown. Statistics for all experiments were performed by two-way ANOVA and post-hoc Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.001. Bars represent mean ± SEM. LPS, lipopolysaccharide; MFI, mean fluorescence intensity; BMDMs, bone marrow-derived macrophages. ns, not significant.

Figure 2

Canonical NF-κB signaling is unaffected by OTULIN haploinsufficiency. (A) Western blotting for p-IκBα and IκBα of LPS-stimulated (5 ng/mL) BMDMs, (representative data from three independent experiments). (B) Western blotting quantification of the three independent experiments. Unpaired Student’s t-test. LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages. ns, not significant.

Figure 3

Otulin ^+/− > Wt chimeras demonstrate a hematopoietic-driven inflammation. (A) Experimental setup. BM, bone marrow; Wt, wild type; litt, littermate. (B) Serum cytokine levels, n = 1 independent experiment, n = 5 mice in each group. (C) Normalized MFI of TNF-α and pro-IL-1β in TNF-α+ or pro-IL-1β+ CD64⁺ monocytes and macrophages to the mean MFI in Wt > Wt litt control mice; pooled data from two independent experiments, n = 4–9 mice in each group. Statistics for all experiments were performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.001. Bars represent mean ± SEM. MFI, mean fluorescence intensity. ns, not significant.

Figure 4

Cell-intrinsic effects contribute to the inflammatory outcome in mixed BM chimera. (A) Experimental setup. BM, bone marrow; Wt, wild type; litt, littermate. (B) Serum cytokine levels, n = 2 independent experiments, n = 9–10 mice in each group. (C) Normalized ratio (fold change) of MFI of TNF-α and pro-IL-1β in TNF-α+ or pro-IL-1β+ CD64⁺ monocytes and macrophages from Otulin^+/− or Wt litt cells to Wt cells; pooled data from three independent experiments, n = 15 mice in each group. Statistics for all experiments were performed by unpaired Student’s t-test. **p < 0.01. Bars represent mean ± SEM. MFI, mean fluorescence intensity. ns, not significant.