spin mice display increased resistance to L. monocytogenes. (A) Bacterial load was visualized in spin homozygotes and C57BL/6J mice 3 days after challenge with 5 × 10⁵ cfu luminescent L. monocytogenes. Visual inspection and quantitation of luminescence demonstrated that spin homozygotes had reduced levels of bacteria compared to controls. (B) C57BL/6J, homozygous spin, or Tnfrsf1a-deficient mice were challenged with 5 × 10⁵ or 10⁶ cfu L. monocytogenes. The bacterial load, depicted as luminescence in photons per second, was determined 2 and 3 days after infection. Tnfrsf1a-deficient mice succumbed within 3 days of infection. One-way ANOVA and post-hoc Student-Newman-Keuls test, P < 0.05 for C57BL/6J vs. spin homozygotes on day 3 after infection with 5 × 10⁵ cfu, and P < 0.05 for spin homozygous or C57BL/6J vs. Tnfrsf1a^−/− mice on day 2 after infection with 10⁶ cfu. (C) TNF production by peritoneal macrophages isolated from C57BL/6J or spin homozygous mice was measured in vitro in response to treatment with the indicated concentrations of TLR3, TLR2/1, or TLR4 ligands (poly I:C, Pam₃CSK₄, or lipopolysaccharide, respectively). TNF production was comparable between C57BL6/J and spin macrophages. The average response of cells from seven C57BL6/J and seven spin homozygous mice is plotted; error bars represent SD. (D) LPS-stimulated pro-IL-1β production, IκB degradation, and phosphorylation of MAP kinases p38, p42, and p44 in bone marrow-derived macrophages from C57BL/6J or spin homozygous mice were compared by Western blotting of lysates made at the indicated timepoints after LPS treatment in vitro. WT and spin macrophages responded similarly to LPS stimulation.

Autoimmune disease depends on MyD88 in spin mice. (A–D) The levels of serum polyclonal IgM (A) or IgG (B), and antichromatin IgM (C) or antichromatin IgG (D) were measured in 4- to 5-month-old mice of various genotypes. Compound homozygosity for Ptpn6^spin and Myd88^poc suppressed the elevated levels of serum immunoglobulins and antichromatin immunoglobulins found in Ptpn6^spin/spin mice. **, one-way ANOVA; P < 0.0001 and posthoc Student–Newman–Keuls test, P < 0.05 for Ptpn6^spin/spinMyd88^poc/+ vs. all other genotypes.

Chronic inflammation and autoimmune disease in homozygous spin mice depends on microbes. (A–D) Haematoxylin/eosin-stained tissue sections from 6- to 20-week-old Ptpn6^spin/spin mice derived and housed in GF conditions (A and C) and then conventionalized into normal SPF conditions (B and D). The feet of conventionalized mice are inflamed, with epidermal ulcerations displaying superficial necrosis and a leukocyte infiltrate (B), whereas GF mice display no foot inflammation (A). The cellularity of the bone marrow is increased in conventionalized mice, with numerous hematopoietic cells and granuloma-like structures (D). The bone marrow of GF mice appears normal (C). Sections are magnified ×20 (A and B) and ×40 (C and D). (E–H) The levels of serum polyclonal IgM (E) or IgG (F) and antichromatin IgM (G) or antichromatin IgG (H) were measured 6–12 weeks after mice housed in GF conditions were conventionalized by introduction into SPF conditions. GF conditions suppressed the elevated levels of immunoglobulins and anti-chromatin immunoglobulins found in Ptpn6^spin/spin mice housed in SPF conditions. **, Student's t test, P < 0.05 for GF Ptpn6^spin/spin mice vs. specific pathogen-free Ptpn6^spin/spin mice.

Chronic inflammation in homozygous spin mutants. Foot lesions develop in spin homozygotes from 6 weeks of age. Two representative foot lesions from spin homozygotes are shown.

Detection of the spin mutation. (A) The mutation was predicted to introduce a HincII restriction enzyme site in genomic DNA from spin mutant mice. Amplification of Ptpn6 genomic DNA followed by HincII restriction enzyme digest confirmed the presence of the mutation. (B) SHP1 protein levels were normal in purified bone marrow-derived macrophages from spin homozygotes. The Western blot shown in Fig. 2D was reprobed with SHP1 antibodies, or tubulin antibodies as a loading control. Timepoints indicate minutes after stimulation with LPS in vitro.

Incidence curve showing age of onset of plantar inflammation in homozygous spin mice with or without null mutations in Il1r1. For difference between Il1r1^+/+ and Il1r1^−/−, P < 0.0001.