Data on IL-10R neutralization-induced chronic colitis in Lipocalin 2 deficient mice on BALB/c background

The data herein is related to the research article entitled “Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis” (Singh et al., 2016) [1] where we have demonstrated that C57BL/6 Lipocalin 2 deficient mice (Lcn2KO) developed chronic colitis upon anti-interleukin-10 receptor (αIL-10R) monoclonal antibody administration. In the present article, we evaluated the susceptibility of BALB/c Lcn2KO mice and their WT littermates to the αIL-10R neutralization-induced chronic colitis. Our data showed that αIL-10R mAb-treated BALB/c Lcn2KO mice exhibited severe chronic colitis (i.e., splenomegaly, colomegaly, colonic pathology, and incidence of rectal prolapse) when compared to WT mice.


a b s t r a c t
The data herein is related to the research article entitled "Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis" (Singh et al., 2016) [1] where we have demonstrated that C57BL/6 Lipocalin 2 deficient mice (Lcn2KO) developed chronic colitis upon antiinterleukin-10 receptor (αIL-10R) monoclonal antibody administration. In the present article, we evaluated the susceptibility of BALB/c Lcn2KO mice and their WT littermates to the αIL-10R neutralization-induced chronic colitis. Our data showed that αIL-10R mAb-treated BALB/c Lcn2KO mice exhibited severe chronic colitis (i.e., splenomegaly, colomegaly, colonic pathology, and incidence of rectal prolapse) when compared to WT mice.
& The data indicate that Lcn2 deficiency predisposes to colitis and this phenotype can be recapitulated in mice on the BALB/c background.
The data support future studies in delineating the role of Lcn2 in conferring mucoprotection.

Data
The dataset of this article provides additional information to Ref. [1], in which we have characterized the increased susceptibility of C57BL/6 lipocalin 2 knockout (Lcn2KO) mice to colitis. Considering that the mouse genetic background can influence colitogenesis [i.e., immune responses of C57BL/6 and BALB/c mice are Th1 and Th2-biased, respectively [2][3][4][5], we herein investigated the susceptibility of BALB/c Lcn2KO mice to interleukin-10 receptor (IL-10R) neutralization-induced chronic colitis. The data presented here elucidate that the robust chronic colitis observed in αIL-10R-treated C57BL/6 Lcn2KO mice [1] can be recapitulated in BALB/c Lcn2KO mice. Specifically, splenomegaly, colomegaly, and elevated serum and colonic inflammation markers were observed in αIL-10R-treated BALB/c Lcn2KO mice when compared to their respective WT control ( Fig. 1A-E).
Remarkably, BALB/c Lcn2KO mice exhibited rectal prolapse, a severe form of colitis, upon IL-10R neutralization (Fig. 1F). Histological analysis-extent of inflammatory cell infiltrate (ICI), epithelial hyperplasia, goblet cell loss, and distorted crypt structure ( Fig. 1G and H)-further established that BALB/c Lcn2KO mice develop a severe chronic colitis, upon IL-10R neutralization, when compared to WT control.

Mice
Lcn2KO mice [6] and their WT littermates on BALB/c background were maintained under specificpathogen-free conditions in the animal house facility at Pennsylvania State University, PA. Mice were housed in cages (max. 5 mice per cage) and fed on chow-control diet ad libitum with unrestricted access to water. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Pennsylvania State University. Gut microbiota composition was analyzed in BALB/c Lcn2KO mice and their WT littermates as described in ref. [1].

IL-10R neutralization-induced chronic colitis
Four weeks old BALB/c Lcn2KO mice and their WT littermates (n ¼4) were administered with four weekly injections (1.0 mg/mouse, intraperitoneally) of anti-mouse αIL-10R mAb (BioXcell). Control mice were administered with the isotype (IgG1) control antibody. Colonic inflammation was examined by monitoring for body weight, fecal occult blood, and diarrhea. At one week after the last injection of αIL-10R mAb or IgG1 control, the mice were euthanized by CO 2 asphyxiation and assessed for standard chronic colitis parameters.

Enzyme-linked immunosorbent assay
Blood samples were collected in a BD Microtainer (Becton Dickinson) via the retro-orbital plexus at euthanasia. Hemolysis-free sera were collected after centrifugation and stored at À80°C until further analysis. Serum amyloid A (SAA) level was analyzed by ELISA according to the manufacturer's (R & D Systems) protocol.

Quantitative reverse-transcription PCR
Total RNA was isolated from colonic tissue using TRI reagent (Sigma) and used to synthesize cDNA using the cDNA Synthesis Kit (Quanta BioSciences). qRT-PCR was performed with the use of SYBR Green Master Mix (Quanta Bio-Sciences) and primers specific for mouse TNF and 36B4 as described in Ref. [1], and read using the Step One Plus Real-Time PCR Q28 System (Applied Biosystems).

Histology
After euthanasia, mouse colons were prepared as Swiss roll, fixed overnight in 10% neutral buffered formalin and stored in 70% ethanol. Colons were processed for paraffin embedding and serial sections (5 mm) were collected and stained with hematoxylin and eosin (H&E) at the Animal Diagnostic Laboratory, PSU. Histologic scoring was performed as described previously [7].

Statistical analysis
Data are presented as means 7SEM. Statistical significance between the groups was calculated using a one-way ANOVA followed by Tukey's multiple comparison test. P o0.05 was considered statistically significant. All statistical analyses were performed with the GraphPad Prism 7.0 program (GraphPad, Inc.).