IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity
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eLife assessment
The effect of helminth infection on vaccination against tuberculosis infection and disease is an important area of study. In this manuscript, the authors build off of a large body of prior data showing that mycobacterial antigens upregulate MINCLE whilst the cytokine IL-4 downregulates MINCLE. As IL-4 is upregulated during Helminth infections, this can antagonize Th1/Th17 responses. By using two different models of helminth infection, the authors demonstrate an organ-specific impairment of Th17 responses in a vaccination setting with a MINCLE-dependent adjuvant. The work is topical and may have important translational implications for patients with tuberculosis and helminth co-infections and/or vaccination regimens for patients with helminth infections. The study will be of interest to individuals studying the convergence of different infectious diseases.
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Abstract
The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6’-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.
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Author Response
Reviewer #1 (Public Review):
The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.
We thank the Reviewer for evaluation of the manuscript and for the comments. Indeed, published studies have shown that helminth infection can impair the response to the BCG vaccine. However, this manuscript shows for the first time that IL-4 and helminth infection impair MINCLE expression in vivo. In addition, it is the first …
Author Response
Reviewer #1 (Public Review):
The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.
We thank the Reviewer for evaluation of the manuscript and for the comments. Indeed, published studies have shown that helminth infection can impair the response to the BCG vaccine. However, this manuscript shows for the first time that IL-4 and helminth infection impair MINCLE expression in vivo. In addition, it is the first report demonstrating a negative effect of helminth infections on the antigen-specific Th1/Th17 response after vaccination with a MINCLE-dependent adjuvant.
Regarding mechanistic insight, we have employed mice deficient in IL-4/IL-13 to determine whether the thwarted Th1/Th17 response is caused by these Th2 cytokines in helminth-infected mice. New Figure 6 in the revised manuscript indeed demonstrates recovery of antigen-specific IFN and IL-17 production in the absence of IL-4/IL-13.
In terms of the in vivo experimental design, it is unclear why the authors chose to administer BCG IP, when the vaccine is given SC (and then based on more recent data, IV could be arguably interesting and relevant). The focus on the peritoneum limits the potential application of these findings to address the important question of the effects of helminth infection on BCG vaccine responses. The ultimate in vivo experiment to be able to demonstrate a physiological relevance of the mechanisms explored here would be to see what the effect was on Mtb infection in the lung.
BCG was injected i.p. to induce upregulation of MINCLE on peritoneal cells and to be able to ask whether IL-4 and/or helminth infection will lead to a down-regulation of MINCLE expression on myeloid cells in vivo. Thus, we were not interested in this context in the adaptive immune response to BCG. Instead, the peritoneal BCG injection provided access to myeloid cells exposed to Th2 immune condition in vivo for analysis of MINCLE protein levels on the surface. As stated in the Discussion section (lines 400-405 in the revised manuscript), detection of MINCLE by flow cytometry from tissue cells is complicated by the loss of cell surface protein during enzymatic organ digestion.
We agree that it would be interesting to study the impact of helminth infection on BCG-induced protection to Mtb challenge infection in the lung. As we have described here the impairment of Th1/Th17 immune responses after immunization with H1/CAF01 that induces protection (Werninghaus et al. 2009 J Exp Med), it would make most sense to perform such challenge infections first in this setting. However, Mtb infection requires a dedicated BSL3 animal facility, we therefore consider such challenge experiments beyond the scope of this manuscript
The authors do report different responses in the spleen and lymphnode, which is interesting, but lines 336-337 accurately point out that compartmentalized overexpression of IL-10 in the spleens but not the lymph nodes has been described in mice with chronic schistosomiasis. Mechanistic insight into this phenomenon was lacking, and the relevance to Mtb infection is still unknown.
We agree that the mechanism for the compartmentalized regulation of adaptive immune differentiation in helminth-infected mice is not clear.
Reviewer #2 (Public Review):
The manuscript entitled "IL-4 and helminth infection downregulate Mincle-dependent macrophage response to mycobacteria and Th17 adjuvanticity" by Schick et al. demonstrate the inhibitory activity of IL-4 and helminth infection on mycobacteria-mediated Th17 immunity. Overall, the authors reported interesting findings with solid data that advance our understanding of CLR function in fungal-bacterial co-infection.
We thank the Reviewer for the appreciation of our study.
Reviewer #3 (Public Review):
The authors first demonstrated in bone marrow-derived macrophages (BMMs) that IL-4 treatment of BMMs led to a significant reduction of BCG- and TDB-induced MINCLE expression (Fig. 1). While IL-4 treatment did not impact BCG phagocytosis by BMMs, it led to a reduced production of the cytokines G-CSF and TNF by BMMs (Fig. 2). In an elegant model using hydrodynamic injection of mini-circle DNA encoding IL-4, the authors show that IL-4 overexpression abrogated the increased MINCLE expression in monocytes upon BCG infection in vivo. Similar findings were observed in a co-infection model with the hookworm Nippostrongylus brasiliensis, where MINCLE expression on inflammatory monocytes from BCG-infected mice was reduced compared to control mice infected only with BCG (Fig. 3). The key findings of the manuscript include the two murine helminth infection models, S. mansoni as a chronic infection, and N. brasiliensis as a transient infection, in both of which the authors showed an organ-specific inhibition of the Th17 response in a vaccination setting with a MINCLE-dependent adjuvant (Fig. 4 and 5).
Data shown in the manuscript represents a major advance over previous studies because for the first time a relation between IL-4 and MINCLE expression and function is demonstrated in vivo in relevant co-infection models. All experiments have been done with care. Appropriate controls have been included and conclusions are largely supported by the data. Future studies in human patients will be needed to determine the clinical relevance of the findings observed in the murine helminth infection models.
We thank the Reviewer for the positive comments and agree that it will be interesting to study the impact of helminth infection on CLR expression and function in human infection and vaccination settings.
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eLife assessment
The effect of helminth infection on vaccination against tuberculosis infection and disease is an important area of study. In this manuscript, the authors build off of a large body of prior data showing that mycobacterial antigens upregulate MINCLE whilst the cytokine IL-4 downregulates MINCLE. As IL-4 is upregulated during Helminth infections, this can antagonize Th1/Th17 responses. By using two different models of helminth infection, the authors demonstrate an organ-specific impairment of Th17 responses in a vaccination setting with a MINCLE-dependent adjuvant. The work is topical and may have important translational implications for patients with tuberculosis and helminth co-infections and/or vaccination regimens for patients with helminth infections. The study will be of interest to individuals studying the …
eLife assessment
The effect of helminth infection on vaccination against tuberculosis infection and disease is an important area of study. In this manuscript, the authors build off of a large body of prior data showing that mycobacterial antigens upregulate MINCLE whilst the cytokine IL-4 downregulates MINCLE. As IL-4 is upregulated during Helminth infections, this can antagonize Th1/Th17 responses. By using two different models of helminth infection, the authors demonstrate an organ-specific impairment of Th17 responses in a vaccination setting with a MINCLE-dependent adjuvant. The work is topical and may have important translational implications for patients with tuberculosis and helminth co-infections and/or vaccination regimens for patients with helminth infections. The study will be of interest to individuals studying the convergence of different infectious diseases.
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Reviewer #1 (Public Review):
The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.
In terms of the in vivo experimental design, it is unclear why the authors chose to administer BCG IP, when the vaccine is given SC (and then based on more recent data, IV could be arguably interesting and relevant). The focus on the peritoneum limits the potential application of these findings to address the important question of the effects of …
Reviewer #1 (Public Review):
The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.
In terms of the in vivo experimental design, it is unclear why the authors chose to administer BCG IP, when the vaccine is given SC (and then based on more recent data, IV could be arguably interesting and relevant). The focus on the peritoneum limits the potential application of these findings to address the important question of the effects of helminth infection on BCG vaccine responses. The ultimate in vivo experiment to be able to demonstrate a physiological relevance of the mechanisms explored here would be to see what the effect was on Mtb infection in the lung.
The authors do report different responses in the spleen and lymphnode, which is interesting, but lines 336-337 accurately point out that compartmentalized overexpression of IL-10 in the spleens but not the lymph nodes has been described in mice with chronic schistosomiasis. Mechanistic insight into this phenomenon was lacking, and the relevance to Mtb infection is still unknown.
-
Reviewer #2 (Public Review):
The manuscript entitled "IL-4 and helminth infection downregulate Mincle-dependent macrophage response to mycobacteria and Th17 adjuvanticity" by Schick et al. demonstrate the inhibitory activity of IL-4 and helminth infection on mycobacteria-mediated Th17 immunity. Overall, the authors reported interesting findings with solid data that advance our understanding of CLR function in fungal-bacterial co-infection.
-
Reviewer #3 (Public Review):
The authors first demonstrated in bone marrow-derived macrophages (BMMs) that IL-4 treatment of BMMs led to a significant reduction of BCG- and TDB-induced MINCLE expression (Fig. 1). While IL-4 treatment did not impact BCG phagocytosis by BMMs, it led to a reduced production of the cytokines G-CSF and TNF by BMMs (Fig. 2). In an elegant model using hydrodynamic injection of mini-circle DNA encoding IL-4, the authors show that IL-4 overexpression abrogated the increased MINCLE expression in monocytes upon BCG infection in vivo. Similar findings were observed in a co-infection model with the hookworm Nippostrongylus brasiliensis, where MINCLE expression on inflammatory monocytes from BCG-infected mice was reduced compared to control mice infected only with BCG (Fig. 3). The key findings of the manuscript …
Reviewer #3 (Public Review):
The authors first demonstrated in bone marrow-derived macrophages (BMMs) that IL-4 treatment of BMMs led to a significant reduction of BCG- and TDB-induced MINCLE expression (Fig. 1). While IL-4 treatment did not impact BCG phagocytosis by BMMs, it led to a reduced production of the cytokines G-CSF and TNF by BMMs (Fig. 2). In an elegant model using hydrodynamic injection of mini-circle DNA encoding IL-4, the authors show that IL-4 overexpression abrogated the increased MINCLE expression in monocytes upon BCG infection in vivo. Similar findings were observed in a co-infection model with the hookworm Nippostrongylus brasiliensis, where MINCLE expression on inflammatory monocytes from BCG-infected mice was reduced compared to control mice infected only with BCG (Fig. 3). The key findings of the manuscript include the two murine helminth infection models, S. mansoni as a chronic infection, and N. brasiliensis as a transient infection, in both of which the authors showed an organ-specific inhibition of the Th17 response in a vaccination setting with a MINCLE-dependent adjuvant (Fig. 4 and 5).
Data shown in the manuscript represents a major advance over previous studies because for the first time a relation between IL-4 and MINCLE expression and function is demonstrated in vivo in relevant co-infection models. All experiments have been done with care. Appropriate controls have been included and conclusions are largely supported by the data. Future studies in human patients will be needed to determine the clinical relevance of the findings observed in the murine helminth infection models.
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