Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation

Abstract

Inflammation has classically been defined histopathologically, especially by the presence of immune cell infiltrates. However, more recent studies suggest a role for "low-grade" inflammation in a variety of disorders ranging from metabolic syndrome to cancer, which is defined by modest elevations in pro-inflammatory gene expression. Consequently, there is a need for cost-effective, non-invasive biomarkers that, ideally, would have the sensitivity to detect low-grade inflammation and have a dynamic range broad enough to reflect classic robust intestinal inflammation. Herein, we report that, for assessment of intestinal inflammation, fecal lipocalin 2 (Lcn-2), measured by ELISA, serves this purpose. Specifically, using a well-characterized mouse model of DSS colitis, we observed that fecal Lcn-2 and intestinal expression of pro-inflammatory cytokines (IL-1β, CXCL1, TNFα) are modestly but significantly induced by very low concentrations of DSS (0.25 and 0.5%), and become markedly elevated at higher concentrations of DSS (1.0 and 4.0%). As expected, careful histopathologic analysis noted only modest immune infiltrates at low DSS concentration and robust colitis at higher DSS concentrations. In accordance, increased levels of the neutrophil product myeloperoxidase (MPO) was only detected in mice given 1.0 and 4.0% DSS. In addition, fecal Lcn-2 marks the severity of spontaneous colitis development in IL-10 deficient mice. Unlike histopathology, MPO, and q-RT-PCR, the assay of fecal Lcn-2 requires only a stool sample, permits measurement over time, and can detect inflammation as early as 1 day following DSS administration. Thus, assay of fecal Lcn-2 by ELISA can function as a non-invasive, sensitive, dynamic, stable and cost-effective means to monitor intestinal inflammation in mice.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. DSS induced gross pathology is dose dependent.

Eight week old male BL6 mice were administered DSS (0, 0.25, 0.5, 1.0, and 4.0%) in drinking water for 7 days and monitored every day (5 mice per group). A, Loss in body weight; B, spleen weight; C, colon length and E, Colon weight. * p<0.05.

Figure 2. DSS dose dependent increase in pro-inflammatory gene expression and histological damages.

Colonic pro-inflammatory cytokine-encoding genes were quantified by qRT-PCR (A, KC; C, TNF-α; D, IL-1β) and KC was quantified in the serum by ELISA (B). Colonic MPO were determined (E), and H & E stained sections of the colon (F) were used for the determination of the histopathological score (G). * p<0.05.

Figure 3. DSS induces Lcn-2 in a dose dependent manner.

Serum Lcn-2 protein levels were analyzed by ELISA (A) and immunoblotting (B). Colonic Lcn-2 mRNA levels were analyzed by qRT-PCR (C) and fecal Lcn-2 levels were measured in feces at day 7 by ELISA (D). *p<0.05.

Figure 4. Substantial elevation of fecal Lcn-2 by DSS, in a time and dose dependent manner.

Fecal Lcn-2 levels were measured in feces from day 0 through 7 by ELISA.

Figure 5. Fecal Lcn-2 marks the spontaneous colitis in IL-10 deficient mice.

WT and IL-10 KO mice (n = 20) were analyzed for colonic MPO (A), H & E stained colon (B) and fecal Lcn-2 (C). Red circle indicate colitic mice.

Figure 6. Substantial decrease in fecal Lcn-2 during mucosal healing.

Mice were given 1.5% DSS in drinking water for 7 days (n = 5) and DSS was discontinued for 29 days. Fecal Lcn-2 levels were measured by ELISA (*p

Figure 7. Fecal Lcn-2 is stable.

Feces from 10 different mice were collected and split in four parts which were stored for 24 h at −20°C, 4°C, or room temperature (RT) or boiled for 1 h. Lcn-2 levels were measured by ELISA and expressed as values relative to those obtained from samples stored at −20°C. (*p