Helminth Coinfection Alters Monocyte Activation, Polarization, and Function in Latent Mycobacterium tuberculosis Infection

Abstract

Helminth infections are known to influence T and B cell responses in latent tuberculosis infection (LTBI). Whether helminth infections also modulate monocyte responses in helminth-LTBI coinfection has not been fully explored. To this end, we examined the activation, polarization, and function of human monocytes isolated from individuals with LTBI with (n = 25) or without (n = 25) coincident Strongyloides stercoralis infection (S. stercoralis-positive and S. stercoralis-negative respectively). Our data reveal that the presence of S. stercoralis infection is associated with lower frequencies of monocytes expressing CD54, CD80, CD86 at baseline (absence of stimulation) and in response to mycobacterial-Ag stimulation than monocytes from S. stercoralis-negative individuals. In contrast, S. stercoralis infection was associated with higher frequencies of M2-like monocytes, as determined by expression of CD206 and CD163. Monocytes from S. stercoralis-positive individuals had a reduced capacity to phagocytose or exhibit respiratory burst activity following mycobacterial-Ag or LPS stimulation and were less capable of expression of IL-1β, TNF-α, IL-6, and IL-12 at baseline and/or following Ag stimulation compared with those without S. stercoralis infection. In addition, definitive treatment of S. stercoralis infection resulted in a significant reversal of the altered monocyte function 6 mo after anthelmintic therapy. Finally, T cells from S. stercoralis-positive individuals exhibited significantly lower activation at baseline or following mycobacterial-Ag stimulation. Therefore, our data highlight the induction of dampened monocyte activation, enhanced M2 polarization, and impaired monocyte function in helminth-LTBI coinfection. Our data also reveal a different mechanism by which helminth infection modulates immune function in LTBI.

Figure.1.. LTBI/Ss coinfection is associated with decreased monocyte activation and increased M2 polarization

Baseline and antigen-stimulated frequencies of monocytes expressing activation and M2 markers were determined by flow cytometry in Ss⁺ (light grey n =15) and Ss⁻ (dark grey n =15) individuals. The (A) baseline (unstimulated) as well as (B) PPD, (C) WCL and (D) P/I stimulated frequencies of monocytes expressing activation markers (CD54, CD80, CD86) and M2 markers (CD163 and CD206) are shown. Each circle represents a single individual and the bars represent the GM values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Mann–Whitney U test.

Figure.2.. LTBI/Ss coinfection is associated with diminished monocyte phagocytosis and respiratory burst activity

Baseline and antigen stimulated frequencies of monocytes were assessed for phagocytic activity and respiratory burst activity in Ss⁺ (light grey n =10) and Ss⁻ (dark grey n =10) individuals. (A) PBMC were stimulated with WCL, LPS and P/I for 6 h and the frequencies of monocytes undergoing phagocytosis was measured. (B) PBMC were stimulated with WCL, LPS and P/I and the frequencies of monocytes undergoing respiratory burst was measured. Each circle represents a single individual and the bars represent the GM values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Mann–Whitney U test.

Figure.3.. LTBI/Ss coinfection is associated with diminished monocyte cytokine production

Baseline and antigen-stimulated frequencies of monocytes expressing cytokines were determined by flow cytometry in Ss⁺ (light grey n =15) and Ss⁻ (dark grey n =15) individuals. The (A) baseline (unstimulated) as well as (B) PPD, (C) WCL and (D) P/I stimulated frequencies of monocytes expressing cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-10 and IL-12) are shown. Each circle represents a single individual and the bars represent the GM values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Mann–Whitney U test.

Figure.4.. Diminished pro-inflammatory cytokine production and enhanced IL-10 production LTBI/Ss coinfection

Baseline and antigen-stimulated levels of pro-inflammatory cytokines were measured by ELISA using PBMC culture supernatants of Ss⁺ (light grey n =15) and Ss⁻ (dark grey n =15) individuals. The (A) baseline (unstimulated) as well as (B) PPD, (C) WCL and (D) P/I stimulated levels of cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-10 and IL-12) are shown. Each circle represents a single individual and the bars represent the GM values. Net cytokine levels were calculated by subtracting baseline cytokine levels from the antigen-stimulated cytokine levels for each individual. The p values were calculated using the Mann–Whitney U test.

Figure.5.. Anthelmintic therapy significantly increases monocyte activation and decreases M2 polarization in LTBI/Ss coinfection

Baseline and antigen-stimulated frequencies of monocytes expressing activation and M2 markers were determined by flow cytometry in Ss⁺ (light grey n =15) individuals before and after treatment with a standard dose of ivermectin and albendazole. The (A) baseline (unstimulated) as well as (B) PPD Ag, (C) WCL and (D) P/I stimulated frequencies of monocyte expressing activation (CD54, CD80, CD86) and M2 (CCR2, CX3CR1) markers are shown. Each line represents a single individual. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Wilcoxon signed rank test.

Figure.6.. Anthelmintic therapy reverses the diminished phagocytosis or respiratory burst activity in monocytes in LTBI/Ss coinfection

Baseline and antigen stimulated frequencies of monocytes were assessed for phagocytic activity and respiratory burst activity by flow cytometry in Ss⁺ (light grey n =10) individuals before and after treatment with a standard dose of ivermectin and albendazole (A) PBMC were stimulated at baseline (unstimulated), WCL, LPS and P/I for 6 h and the frequencies of monocytes undergoing phagocytosis was measured. (B) PBMC were stimulated at baseline (unstimulated), WCL, LPS and P/I for 6 h and the frequencies of monocytes undergoing respiratory burst activity was measured. Each line represents a single individual. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Wilcoxon signed rank test.

Figure.7.. Elevated levels of monocytes producing cytokines in LTBI/Ss co-infection following anthelmintic therapy

Baseline and antigen-stimulated frequencies of monocytes expressing cytokines were determined by flow cytometry in Ss⁺ (light grey n =15) individuals before and after treatment with a standard dose of ivermectin and albendazole. The (A) baseline (unstimulated) as well as (B) PPD, (C) WCL and (D) P/I stimulated frequencies of monocytes expressing cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-10 and IL-12) were measured. Each line represents a single individual. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Wilcoxon signed rank test.

Figure.8.. Anthelmintic therapy significantly increases pro-inflammatory cytokine production in LTBI/Ss co-infection

Baseline and antigen-stimulated levels of pro-inflammatory cytokines were determined by ELISA using PBMC culture supernatants of Ss⁺ (light grey n =15) individuals before and after treatment with a standard dose of ivermectin and albendazole. The (A) baseline (unstimulated) as well as (B) PPD, (C) WCL and (D) P/I stimulated levels of cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-10 and IL-12) were measured. Each line represents a single individual. Net cytokine levels were calculated by subtracting baseline cytokine levels from the antigen-induced cytokine levels for each individual. The p values were calculated using the Wilcoxon signed rank test.

Figure.9.. LTBI/Ss coinfection is associated with decreased T cell activation

Baseline and antigen-stimulated frequencies of T cell activation markers were determined by flow cytometry in Ss⁺ (light grey n =15) and Ss⁻ (dark grey n =15) individuals. (A) Baseline (unstimulated), PPD Ag, WCL and P/I stimulated frequencies of T cell activation markers (CD69 and HLA-DR) expressed by CD4⁺ T cells are shown. (B) Baseline (unstimulated), PPD Ag, WCL and P/I stimulated frequencies of T cell activation markers (CD69 and HLA-DR) expressed by CD8⁺ T cells are shown. Each circle represents a single individual and the bars represent the GM values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. The p values were calculated using the Mann–Whitney U test.