Alternative mechanisms for controlling chronic parasite infection associated with experimental malaria

        Recent Center for Disease Control data from 2016 estimated around 216 million cases and 445,000 deaths attributed to Malaria, mainly concentrated in Sub-Saharan African regions (1).  The parasite Plasmodium is transmitted by mosquitoes and the infection can be fatal if the immune system cannot mount the appropriate response.  There are several species of Plasmodium; P. chabaudi is commonly used in experimental studies in mice and P. falciparum is one of the common species infecting humans (2).  P. chabaudi is studied in experimental malaria due to similarities to P. falciparum (2).  A new study published by Borges da Silva et al (2018) studied a potential alternative mechanism for controlling the chronic parasite levels associated with experimental malaria (3).

         The classical response to infection by plasmodium parasite relies on IgG antibodies and the creation of memory T and B cells in the adaptive response (2,3).  Clinical immunity and increased resistance to the parasite can develop with constant exposure to the same strain over time.  Once infected, it is important to keep chronic parasite levels low or to eliminate the parasite entirely.  Previous research has shown that CD28, a cluster of cell surface proteins that are important for stimulating helper T cells, are necessary to completely eliminate parasite levels (3).  This shows the classical requirement of helper T cells for activating production of IgG antibodies and long lasting memory cells that assist in developing clinical immunity.  The researchers discovered that mice without CD28 were still able to maintain low levels of parasitemia and investigated the alternative mechanism by which this is possible.  The study eliminated CD28 from mice and measured the comparative levels of detectable parasite in the blood after primary and secondary infections, finding that over time, CD28KO mice could produce long lasting immunity just like the wild type mice (Figure 1). 

 Figure 1a,c,d. Parasitemia levels in C57BL/6 and CD28KO mice following primary and secondary infections. (a) Mice lacking CD28 co-stimulatory molecule could still control parasite levels upon initial infection of iRBCs. (c,d) Following reinfection at 40 and 80 days, previously infected C57BL/6 and CD28KO mice could control the secondary response to parasites better than controls.  

Typically, IgM antibodies are involved in the first wave in the immune response, but higher affinity antibodies like IgG take control of the response as it continues and persist as memory B cells for re-infection and secondary responses.  To switch from IgM to IgG antibodies, helper T cells are required.  By knocking out or eliminating CD28, the ability of helper T cells to stimulate the switch of antibody type was eliminated.  Figure 2 shows the changes in IgM levels in both types of mice.  The wild type mice had an initial peak of IgM serum levels related to the first wave, but that decreased as IgG antibodies increased in concentration to sustain immunity.  The CD28KO mice increased IgM levels consistently over time.  The researchers discovered that the persistence of IgM was due to the mechanism of controlling chronic infection because after curing the CD28KO mice with chloroquine treatment, their total levels of anti-parasite IgM decreased (3).

 Figure 2a-c. IgM and IgG levels in C57BL/6 and CD28KO mice following initial Pc-iRBC infection. (a-c) C57BL/6 mice experience initial primary increase in IgM antibodies during immediate response but increase IgG concentration over time.  CD28KO mice gradually increase IgM concentrations until greater than C57BL/6 levels and do not show changes in IgG serum levels.

These mice lacked the ability to produce traditional memory cells, so researchers investigated the contents of the spleen and discovered IgM+ experienced B cells. B cells gather in the spleen, specifically in germinal centers to undergo isotype switching and affinity maturation processes that lead to higher affinity and more specific antibody production.  Researchers found that the spleens of mice lacking CD28 weighed more than those of wild type mice and had increased numbers of CD19+ (splenic B) and IgM CD138+ plasmocytes.  A key marker of germinal center B cells is Fas+GL7+, and there were very few cells expressing this marker in CD28KO mice, suggesting that any memory cells created would be a result of a germinal center-independent mechanism (3, 5).  Interestingly, at 100 days after infection, researchers discovered IgM+Fas+GL7- B cells that expressed CD38, a marker for memory B cells.  These cells, called IgM+ experienced cells, were found under conditions of chronic and residual parasite infection and would have developed independently from germinal centers.  Following in vitro stimulation, IgM+ experienced cells could proliferate and differentiate to produce plasmocytes and IgM in response to parasite infection (Figure 4).  More information about IgM Memory B cells can be found here to understand their historical significance, signaling, and relevance in current immune responses (4).   

Figure 4e, j. Splenic B cell populations in C57BL/6 and CD28KO mice during chronic infection. (e) Higher numbers of Fas+GL7- splenic B cells in CD28KO mice. (j). CQ is chloroquine treatment to eliminate infection. Larger IgM+ experienced splenic B cell population (Fas+GL7-CD38+CD73-) in chronically infected CD28KO mice compared to cured CD28KO and C57BL/6.

Finally, the researchers confirmed that the IgM antibodies were responsible for this mechanism of controlling parasite infection by testing that they recognized infected red blood cells.  The CD28KO antibodies mediated complement activation and lysis of infected cells, and showed that dendritic and macrophages were able to present infected red blood cells as antigens on their surfaces.  IgM antibodies from the CD28KO mice 100 days after infection were compared with 15-day post infection wild type mice and showed similar recognition of infected red blood cells.  The IgM antibodies from the CD28KO mice could transfer protection and were effective, whereas IgM from wild type mice 100 days after infection were not effective at activating complement, lysis, and increasing infected red blood cell uptake by antigen presenting cells (3).  This shows that the IgM+ experienced B cells are unique and provide an effective control of parasite infection.

         Overall, researchers identified that IgM+ experienced B cells could effectively stimulate immune responses to control parasite levels via an alternative mechanism without stimulation of helper T cells to generate classical memory cells.  This mechanism that was investigated and evaluated in this study raises several future questions.  Since this is an alternative mechanism, the role of IgM+ experienced cells when CD28 and classical memory cell development is functional is unclear. Several of the experiments done by Borgas da Silva et al did not show increased levels of IgM during chronic infection in cells with functional CD28, raising questions about how important this mechanism is in traditionally functioning cells.  The IgM response could be a primitive first line defense, or it could have implications for being a backup response in cases of immunodeficiency (3,4).  The relevance of this research towards human immunity in malaria patients is unknown and deserves future investigation.  It would be important to investigate whether IgM plays a protective and significant role in the chronic immune responses to malaria, specifically in endemic areas, with relevance to the creation of developing a vaccine (3). 

Sources:

1. Centers for Disease Control and Prevention (2018, October 9). Parasites- Malaria. Retrieved from https://www.cdc.gov/parasites/malaria/index.html. 

2. Stephens, R., Culleton, R.L., & Lamb, T. J. (2012).  The contribution of Plasmodium chabaudi to our understanding of malaria. Trends in Parasitology, 28(2), 73-82. doi:10.1016/j.pt.2011.10.006.

3. Borges da Silva, H., Machado de Salles, É. Lima-Mauro, E.F., Sardinha, L.R., Álvarez, J. M., & D’Império Lima, M.R. (2018). CD28 Deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria. PLoS ONE, 13(8). https://doi.org/10.1371/journal.pone.0202522

4. Capolunghi, F. Roasado, M.M, Sinibaldi, M. Aranburu, A. & Carsetti, R. (2013). Why do we need IgM memory B cells? Immunology Letters, 152, 114-120.  https://doi.org/10.1016/j.imlet.2013.04.007.

5. Nojima, T., Haniuda, K, Moutai, T, Matsudaira, M, Mizokawa, S., Shiratori, I., Azuma, T. & Kitamura, D. (2011). In-vitro derived germinal center B cells differentially generate memory B or plasma cells in vivo. Nature Communications, 2(465). doi: 10.1038/ncomms1475.

6. Schofield, L., & Mueller, I. (2006). Clinical Immunity to Malaria. Current Molecular Medicine, 6(2). doi: 10.2174/156652406776055221