B Cells Protect T Cell-deficient mice from Cryptococcal Brain Invasion

KEYWORDS:

• brain
• B cells
• cryptococcus
• IgM
• T cells

This article refers to:

Naïve B cells reduce fungal dissemination in Cryptococcus neoformans infected Rag1−/− mice

In this issue of Virulence, Dufaud et al¹ utilize a Rag1-deficient murine model and adoptive transfers of B-cells or IgM-containing serum to directly examine the role of B-cells in host defense against the opportunistic yeast fungus Cryptococcus neoformans (CN). Cryptococcal meningoencephalitis remains a leading cause of mortality in patients with HIV/AIDS resulting in ∼200,000 annual deaths worldwide,² hence, better understanding of the cellular and molecular factors that protect the host from cryptococcosis could lead to immune-based therapies and better patient outcomes. The susceptibility of patients with AIDS and idiopathic CD4+ T-cell lymphocytopenia to cryptococcosis clearly underscores the critical contribution of CD4+ T-cells in effective host defense. Moreover, macrophages are critical in promoting fungal clearance following their priming and effective cross-talk with T-cells via the production of IFN-gamma.³ On the other hand, the role of B-cells in anti-CN host defense has remained less understood. Prior studies in C57BL/6 mice depleted of B-1 cells,⁴ in sIgM^−/− mice that lack secreted IgM,⁵ and in X-linked immunodeficient mice (XID) that possess a mutation in the Bruton's tyrosine kinase (Btk),⁶ have suggested a contribution of B-1 cells or naïve serum IgM in protection against cryptococcal brain invasion; however, definitive conclusions about the direct role of B-cells in anti-CN immunity were confounded in all of the aforementioned studies by the presence of T cells and because of associated defects in cellular immunity (XID mice) and in B-cell development (sIgM^−/− mice).

In the present study, the authors utilize a low-virulence strain of CN, which upon infection of C57BL/6 mice results in a significant chronic pulmonary infection with only low-level dissemination and efficient local control of the fungus in the brain.⁷ In contrast to C57BL/6 mice, Dufaud el al show that CN-infected Rag1-deficient mice (on the C57BL/6 genetic background), which lack both T and B-cells, display a marked increase in fungal proliferation in the brain, which is particularly accentuated after the second week post-infection (Figure 1B). The authors restore (at least partly) B-cell function in Rag1-deficient hosts by adoptively transferring B-cells purified from the spleens of C57BL/6 mice into the Rag1-deficient animals. This transfer restored the numbers of B-1a and B-1b cells recruited to the CN-infected lungs, but not T-cells or B-2 cells, in agreement with previous reports of B-cell adoptive transfers in other inflammatory settings.⁸ Intriguingly, this B-cell transfer led to a marked reduction in the burden of CN in the brain of these mice compared to Rag1-deficient mice that did receive B-cells, a reduction in fungal load that reached levels comparable to those seen in CN-infected C57BL/6 mice. Instead, B-cell adoptive transfers in Rag1-deficient mice did not affect the fungal load in the CN-infected lung, highlighting a brain-specific requirement for B cell-dependent control of fungal load during cryptococcosis. This brain-specific role of B-cells in protection against cryptococcosis is is on par with recent clinical reports of brain infections by CN (and other fungi including Aspergillus) in patients receiving the small-molecule irreversible BTK inhibitor ibrutinib, which impairs B-cell receptor signaling, although patients with inherited BTK deficiency have not been reported to develop brain cryptococcal disease thus far.^9–10

The results presented by Dufaud and colleagues are the most definitive data to date linking B-cells and cryptococcal host defense in the brain, especially in light of the demonstrated ability of B-cells to promote protection despite the absence of T-cells. Yet, important questions remain with regard to the mechanism(s) by which the transferred B-cells exert their protective effects and reduced CN invasion in the brain. While B-cells are best known for their secretion of antibody, they also participate in immune signaling, secrete inflammatory mediators, phagocytose and kill microbes and present antigen to T-cells.^11–13 With the exception of the latter mechanism which is not pertinent herein given that the results were obtained in Rag1-deficient mice that lack T-cells, all other mechanisms remain viable possibilities. Dufaud et al attempted to determine if the observed B cell-mediated protection was due to secreted antibody or due to other B cell-intrinsic activities. They found that adoptive transfer of and restoration of B-cells in the lungs of CN-infected Rag1-deficient mice dramatically increased the levels of IgG and IgM in Rag1-deficient mice which otherwise have undetectable levels of antibody in their serum. However, this antibody was not necessarily CN-specific as these antibodies did not recognize GXM, the major CN-capsule antigen; future work will be needed to determine whether these antibodies recognize other major CN antigens.

Notably, transfer of IgG-depleted, IgM-containing serum in Rag1-deficient hosts resulted in an increase in alveolar macrophage phagocytic index compared to Rag1-deficient mice that received serum lacking both IgG and IgM. No information was provided within this work on whether the IgM-containing serum transfer restored brain control of CN; this will be an important question to be answered in future studies. From these data, the authors suggested a potential role of transferred B-1 cells via secretion of IgM in increasing macrophage uptake of CN as a potential mechanism mediating B cell-dependent protection from CN invasion. Whether uptake by alveolar macrophages represents a conclusive readout for protecting against CN invasion is unclear. Recruited M1 polarized monocyte-derived macrophages are thought to be microbicidal effector cells (often termed inflammatory macrophages “iMac” or exudate macrophages “exMac”), whereas alveolar macrophages have very limited microbicidal activity.¹⁴ This may be particularly important as non-fungicidal macrophages have been implicated in promoting, instead of restricting, CN dissemination outside the lungs.^15–16

Therefore, future studies will be required to examine additional measures of lung immunity and to study brain-specific correlates of protection in Rag1-deficient mice following transfer of IgM-containing serum or B-cells in order to further discern the B cell-intrinsic versus antibody-dependent mechanisms of protection. In fact, the results of enhanced pro-inflammatory cytokine production and preserved granulomatous architecture in the CN-infected lungs of some Rag1-deficient mice after B-cell adoptive transfer indicates that a broader investigation of cytokine/chemokine production and of immune cell recruitment may shed further light into the role of B-cells beyond antibody secretion in accounting for the observed protection against CN invasion. Importantly, the inability of Rag1-deficient mice to control fungal proliferation locally in the brain between weeks 2 and 5 post-infection points (Figure 1B) toward a role of lymphocytes (including B-cells) in priming local brain immune responses via microglial activation, not just in preventing dissemination from the lungs into the brain. Hence, future studies will be useful to determine the impact of B-cell or IgM-containing serum transfers on local brain anti-CN host responses by microglial cells. Lastly, it will be of interest to determine whether B-cells protect against lung and brain invasion following infection with other CN strains of different molecular types and with Cryptococcus gattii, which has a propensity for pulmonary, not brain, disease in mice and humans.¹⁷

In summary, the study by Dufaud and colleagues provides the most direct evidence thus far regarding the role of B-cells in controlling CN invasion of the brain. This important study paves the path for additional mechanistic studies that will shed further light on the tissue-specific and antibody-dependent and –independent roles of B-cells in antifungal immunity. These studies may collectively help to design B cell-dependent therapeutic strategies to combat this devastating infection, which based on the results of the present study may be amenable and effective even in patients who lack CD4+ T-cells, that is, those at the highest risk of developing cryptococcosis.

Acknowledgements

This work was supported by the Division of Intramural Research of the NIAID, NIH.

Additional information

Funding

Intramural Research Program, NIAID ID: NIH