Gastrointestinal Helminth Infection Improves Insulin Sensitivity, Decreases Systemic Inflammation, and Alters the Composition of Gut Microbiota in Distinct Mouse Models of Type 2 Diabetes

Abstract

Type 2 diabetes (T2D) is a major health problem and is considered one of the top 10 diseases leading to death globally. T2D has been widely associated with systemic and local inflammatory responses and with alterations in the gut microbiota. Microorganisms, including parasitic worms and gut microbes have exquisitely co-evolved with their hosts to establish an immunological interaction that is essential for the formation and maintenance of a balanced immune system, including suppression of excessive inflammation. Herein we show that both prophylactic and therapeutic infection of mice with the parasitic hookworm-like nematode, Nippostrongylus brasiliensis, significantly reduced fasting blood glucose, oral glucose tolerance and body weight gain in two different diet-induced mouse models of T2D. Helminth infection was associated with elevated type 2 immune responses including increased eosinophil numbers in the mesenteric lymph nodes, liver and adipose tissues, as well as increased expression of IL-4 and alternatively activated macrophage marker genes in adipose tissue, liver and gut. N. brasiliensis infection was also associated with significant compositional changes in the gut microbiota at both the phylum and order levels. Our findings show that N. brasiliensis infection drives changes in local and systemic immune cell populations, and that these changes are associated with a reduction in systemic and local inflammation and compositional changes in the gut microbiota which cumulatively might be responsible for the improved insulin sensitivity observed in infected mice. Our findings indicate that carefully controlled therapeutic hookworm infection in humans could be a novel approach for treating metabolic syndrome and thereby preventing T2D.

Keywords: M2 macrophages; Nippostrongylus brasiliensis; eosinophils; helminth; high fat diet; high glycemic index diet; microbiota; type 2 diabetes.

Figure 1

Experimental design (A) and timeline for infection of mice with Nippostrongylus brasiliensis third stage larvae (L3) (B). NC, Normal Control diet; HGI, High Glycemic Index diet; HF, High Fat diet.

Figure 2

Nippostrongylus brasiliensis infection decreased fasting blood glucose (FBG) and improved glucose metabolism in high glycaemic index (HGI) and high fat (HF) diet models of type 2 diabetes. C57BL/6 mice were fed normal control (NC), HF or HGI diet and infected once monthly with 500 infective larvae of N. brasiliensis commencing at 6 weeks of age for prophylactic infections and 24 weeks of age for therapeutic infections. (A) FBG in mice fed on different diets and administered prophylactic infection with N. brasiliensis. (B) FBG in mice fed on different diets and administered therapeutic infection with N. brasiliensis. Oral glucose tolerance test (OGTT) in mice fed on NC or HGI diets and administered prophylactic (C) or therapeutic (D) infection with N. brasiliensis. Oral glucose tolerance test (OGTT) in mice fed on NC or HF diets and administered prophylactic (E) or therapeutic (F) infection with N. brasiliensis. Area under the curve (AUC) in mice fed on different diets and administered prophylactic (G) or therapeutic (H) infection with N. brasiliensis. Statistical significance was determined with Student’s t test or Two-way analysis of variance (ANOVA). Data are expressed as means ± SEM or means ± SD are representative of two experiments where n = 5/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

Nippostrongylus brasiliensis infection reduced weight gain in high glycemic index (HGI) and high fat (HF) diet models of T2D. C57BL/6 mice were fed normal control (NC), HF, or HGI diet and infected once monthly with 500 infective larvae of N. brasiliensis commencing at 6 weeks of age for prophylactic infections and 24 weeks of age for therapeutic infections. (A) Body weight of mice fed on HGI diet and administered prophylactic infection with N. brasiliensis (Nb). (B) Body weight of mice fed on HGI diet and administered therapeutic infection with Nb. (C) Body weight of mice fed on HF diet and administered prophylactic infection with Nb. (D) Body weight of mice fed on HF diet and administered therapeutic infection with Nb. Statistical significance was determined with Two-way analysis of variance (ANOVA). Data are expressed as mean ± SD and are representative of two experiments where n = 5/group. *p < 0.05; **p < 0.01.

Figure 4

Increase in the frequency of eosinophils in the mesenteric lymph nodes (MLN), adipose tissue (AT), liver, and duodenum in mice fed on different diets and infected or not with Nippostrongylus brasiliensis. C57BL/6 mice were fed normal control (NC), high fat (HF), or high glycemic index (HGI) diet and infected once monthly with 500 N. brasiliensis infective larvae from 6 weeks of age [prophylactic, panel (A)] or 24 weeks of age [therapeutic, panel (B)]. Eosinophil frequency and total numbers in MLN, AT, and liver are shown. Eosinophil numbers per high power field (HPF) (magnification x40) in the gut are shown in panel (C) (prophylactic) and panel (D) (therapeutic). Statistical significance was determined with Student’s t test. Data are expressed as mean ± SEM and are representative of two experiments where n = 5/group. **p < 0.01.

Figure 5

Increased expression of Il4 in adipose tissue (AT), liver and gut of mice fed on different diets and infected with Nippostrongylus brasiliensis compared to uninfected mice. C57BL/6 mice were fed normal control (NC), high fat (HF), or high glycemic index (HGI) diet and infected once monthly with 500 N. brasiliensis infective larvae from 6 weeks of age [prophylactic, panel (A)] or 24 weeks of age [therapeutic, panel (B)]. Statistical significance was determined with Student’s t test. Data are expressed as mean ± SEM and are representative of two experiments where n = 5/group. *p < 0.05; **p < 0.01.

Figure 6

Increased expression of Retnla and Chil3 genes in adipose tissue (AT), liver, and duodenum of mice fed on different diets and infected with Nippostrongylus brasiliensis compared to uninfected mice. C57BL/6 mice were fed normal control (NC), high fat (HF), or high glycemic index (HGI) diet and infected once monthly with 500 N. brasiliensis infective larvae from 6 weeks of age [prophylactic, panel (A)] or 24 weeks of age [therapeutic, panel (B)]. Data are expressed as mean ± SEM and are representative of two experiments where n = 5/group. *p < 0.05; **p < 0.01.

Figure 7

Multivariate analysis of differences in the microbial profiles in the small intestine of Nippostrongylus brasiliensis (Nb) infected and uninfected (naïve, N) C57BL/6 mice fed on normal control (NC), high fat (HF), or high glycaemic index (HGI) diet (A). Relative abundance of bacterial phyla in the small intestine of Nb and N mice fed on NC, HF, or HGI diet (B) Relative abundance of bacterial orders in the small intestine of Nb and N mice fed on NC, HF, or HGI diets (C), and abundance of defined taxa where significant differences between infected and uninfected groups were detected (D, E). Mice were infected once monthly from 6 weeks of age with Nb infective larvae. P values are based on ANOVA-like differential expression analysis and are representative of two experiments where n = 5/group. *p < 0.05; **p < 0.01.