Effects of myeloperoxidase on inflammatory responses with hypoxia in Citrobacter rodentium-infectious mice

Abstract

Purpose: Myeloperoxidase (MPO) has been identified as a mediator in various inflammatory diseases. Bacterial infection of the intestine and hypoxia can both lead to inflammatory responses, but the role of MPO in these phenomena remains unclear.

Methods: By building the MPO^-/- mice, we evaluated relevant inflammatory factors and tissue damage in mice with intestinal Citrobacter rodentium infection and hypoxia. The body weight and excreted microorganisms were monitored. Intestinal tissues were collected 7 days after bacterial infection under hypoxia to undergo haematoxylin-eosin staining and assess the degree of pathological damage. ELISA assays were performed to quantify the serum levels of TNF-α, IFN-γ, IL-6, and IL-1β inflammatory cytokines. PCR, WB, and IF assays were conducted to determine the expression of chemokines MCP1, MIP2, and KC in the colon and spleen.

Results: The C. rodentium infection and hypoxia caused weight loss, intestinal colitis, and splenic inflammatory cells active proliferation in wild-type mice. MPO deficiency alleviated this phenomenon. MPO^-/- mice also displayed a significant decline in bacteria clearing ability. The level of TNF-α in the serum and spleen was both lower in MPO^-/- hypoxia C. rodentium-infected mice than that in wild-type mice. The chemokines expression levels of MIP2, KC, and MCP1 in the spleen and colon of each bacterial infected group were significantly increased (p < .05), while in hypoxia, the factors in the spleen and colon were decreased (p < .05). MPO deficiency was found to lower the levels of these chemokines compared with wild-type mice.

Conclusion: MPO plays an important role of the inflammatory responses in infectious enteritis and hypoxia in mice, and the loss of MPO may greatly reduce the body's inflammatory responses to fight diseases.

Keywords: MPO; colitis; hypoxia; inflammatory responses.

Figure 1

Changes in the body weight of mice in each group. The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 2

(A) Twenty‐four hours results of mouse fecal smear culture. (B) The number of bacteria excreted by mice in each group. The WT Citrobacter rodentium‐infection group (NI), MPO^‐/‐ C. rodentium‐infection group (KI), WT hypoxia C. rodentium‐infected group (HI), and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 3

(A) Photographs of the colons of mice in each group. (B) Changes in the colon length of mice in each group. The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 4

(A) Pathological sections of the colons of mice in each group (200×). (B) Results of pathological scoring of mice in each group. (C) Histopathological sections of the spleens of mice in each group (200×). The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 5

(A) Detection of related inflammatory factors in serum by ELISA. (B) Detection of related inflammatory factors in the spleen by ELISA. (C) Detection of inflammatory factors in the colon by PCR. The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 6

(A) Detection of phagocytic chemokines in the spleen by PCR. (B) Detection of phagocytic chemokines in the colon. (C) Detection of related chemokines in the spleen by WB. (D) Detection of related chemokines in the colon by WB. The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI).

Figure 7

Results of immunofluorescence staining for chemokines in the spleen and colon of mice in each group. The WT normoxia control group (NC), MPO^‐/‐ normoxia control group (KC), WT Citrobacter rodentium infection group (NI), MPO^‐/‐ C. rodentium infection group (KI), WT hypoxia control group (HC), MPO^‐/‐ hypoxia control group (HKC), WT hypoxia C. rodentium‐infected group (HI) and MPO^‐/‐ hypoxia C. rodentium‐infected group (HKI). (A) MIP2 for WT‐spleen. (B) MIP2 for MPO^‐/‐ spleen. (C) KC for WT‐spleen. (D) KC for MPO^‐/‐ spleen. (E) MCP1 for WT‐spleen. (F) MCP1 for MPO^‐/‐ spleen. (G) MIP2 for WT‐colon. (H) MIP2 for MPO^‐/‐ colon. (I) KC for WT‐colon. (J) KC for MPO^‐/‐ colon. (K) MCP1 for WT‐colon. (L) MCP1 for MPO^‐/‐ colon.