Primed innate immunity leads to autoinflammatory disease in PSTPIP2-deficient cmo mice

Abstract

The mouse Lupo (I282N) mutation in proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) leads to reduced expression of PSTPIP2 that is associated with a macrophage-mediated autoinflammatory disease. Another mutation in PSTPIP2, L98P, termed chronic multifocal osteomyelits (cmo), leads to a disease in mice that resembles chronic recurrent multifocal osteomyelits in humans. The cellular basis of cmo disease was investigated. cmo disease develops independently of lymphocytes and is cured by bone marrow transplantation. Macrophages, mast cells, and osteoclasts from cmo mice fail to express detectable PSTPIP2 protein. Asymptomatic Pstpip2(cmo/cmo) mice have increased circulating levels of macrophage inflammatory protein 1-alpha and interleukin-6, and their macrophages exhibit increased production of these inflammatory mediators, which is normalized by retroviral expression of wild-type PSTPIP2. Spleens of asymptomatic cmo mice contain increased numbers of macrophage precursors, and cmo mice mobilize more macrophage precursors in response to a sterile inflammatory stimulus. Signal transducer and activator of transcription 1 is elevated in cmo splenic macrophages, which also exhibit increased colony-stimulating factor-1-stimulated proliferation and increased extracellular signal-regulated kinase 1/2 phosphorylation. PSTPIP2 overexpression in macrophages leads to the opposite phenotype. Thus, PSTPIP2 deficiency causes both an expansion of macrophage progenitors and increased responsiveness of mature macrophages to activating stimuli, which together prime the organism for exaggerated and sustained responses leading to autoinflammatory disease.

Figure 1

PSTPIP2 deficiency in macrophages, mast cells, and osteoclasts of cmo mice is associated with splenomegaly and increased numbers of macrophages in the spleen and tails of affected cmo mice. (A) Western blots (WB) of whole-cell lysates of bone marrow–derived macrophages (BMM), mast cells (MC), and osteoclasts (OC) obtained from wt and cmo mice probed with an antibody against PSTPIP2. (B) Absence of PSTPIP2 in immunoprecipitates (IP) from cmo BMM. IgG indicates immunoglobulin G. (C) Sections of tails and spleens of cmo mice stained for the macrophage marker F4/80 (brown) and counterstained with hematoxylin (blue) showing extensive macrophage infiltration. (D) Development of splenomegaly in cmo mice more than 13 weeks old. Data ± SD, *P < .01, Student t test; n ≥ 10.

Figure 2

Increased levels of inflammatory mediators are produced by macrophages in cmo mice before the onset of overt disease. (A) Secretion of cytokines and chemokines by cultured BMM from wt and cmo mice (n ≥ 5). (B) LPS-stimulated inflammatory mediator release by BMM of wt and cmo mice (n ≥ 5). (C) Reconstitution of PSTPIP2 expression in cmo macrophages by retroviral transduction. The vertical line indicates a repositioned gel lane from the same blot. (D) Reconstitution of PSTPIP2 expression in immortalized cmo BMM restores normal LPS-stimulated production of IL-6 and MIP-1α. (E) Screening of cytokine and chemokine levels in mouse sera from asymptomatic (6-8 weeks of age, n = 10-27) and diseased (20 weeks of age, n = 8-11) cmo mice and wt control mice. Data ± SD. *P < .05, †P < .01, Student t test. No significant differences in serum levels or BMM secretion were found for the other inflammatory mediators tested (BLC, D30L, Ltaxin, Eotaxin-2, Fas ligand, Fractalkine, G-CSF, GM-CSF, M-CSF, IFN-γ, IL-2, IL-3, IL-4, IL-9, IL-10, IL-12p40p70, IL-12p70, IL-13, IL-17, I-TAC, KC, Leptin, LIX, Lymphotactin, MIG, MIP-1γ, RANTES, SDF-1, TCA-3, TECK, TIMP-1, TIMP-2, sTNFRI, and sTNFRII).

Figure 3

Expansion of myeloid progenitors in cmo mice precedes disease onset. (A) Increased frequency of CFU-M in spleens of asymptomatic and diseased cmo mice. (B) Splenic cellularity increases after disease onset. (C) Increased frequency of Mac1⁺ WGA⁺ Ly6C^hi late monocyte precursors in the BM of cmo mice. Data ± SD, n ≥ 3 (A-B), n ≥ 10 (C); *P < .05 versus wt, †P < .05 versus cmo asymptomatic.

Figure 4

PSTPIP2 attenuates macrophage proliferation. Proliferation of nonadherent splenocytes (A) or nonadherent BM cells (B) from wt and asymptomatic cmo mice. Triplicate samples with cells harvested from 2 mice/genotype. (C) Growth curves of BAC1.2F5 macrophages retrovirally transduced with MSCV-IRES-GFP vector (empty vector) or MSCV-IRES-GFP-PSTPIP2 (PSTPIP2 OE). The doubling times are presented on the right side of each curve. Data ± SD, n = 3; *P < .01 versus wt, †P < .05 versus wt.

Figure 5

PSTPIP2 negatively regulates STAT1 expression and the activation of Erk1/2 and STAT1 by c-fms in splenic macrophages. (A) CSF-1 and IL-6 signaling in primary SDM from wt and cmo mice. The vertical lines indicate repositioned gel lanes from the same blot. (B) PSTPIP2 overexpression in BAC1.2F5 macrophages inhibits Erk1/2 activation and STAT1 expression. These results were reproduced in 3 to 5 experiments.

Figure 6

Increased thioglycollate-induced recruitment of macrophage precursors in peritoneal exudates of cmo mice. (A) Both wt and cmo mice recruit comparable numbers of leukocytes in the inflamed peritoneum. (B) Phenotypic analysis of the leukocytes in peritoneal exudates. Data ± SD, n = 7; *P < .01 versus wt.

Figure 7

A hypothetic model of the mechanism by which PSTPIP2 deficiency in cmo mice primes the innate immune system for exaggerated and prolonged inflammatory responses. Before disease onset (solid lines), cmo mice produce increased numbers of primitive myeloid progenitors (HPP-CFC) in the BM that migrate to the spleen, where they either become resident HPP-CFCs or differentiate into CFU-M. Tissue damage (dashed lines) induces increased production of MIP-1α and IL-6 by tissue macrophages. IL-6 promotes the proliferation of splenic HPP-CFC, and MIP-1α increases the recruitment of circulating (pro-) monocytes to peripheral tissues (), thus establishing a positive feedback loop. MIP-1α and IL-6 also promote the recruitment of osteoclast precursors and osteoclastogenesis, leading to inflammatory bone resorption.