We previously found that P. chabaudi
infection of mice induced robust expression of an interferon-induced gene signature as the earliest detectable expression response in blood 
. Here, we demonstrate that this ISG response is the combined result of T1IFNs and IFNG, acting in a largely redundant fashion. Although the prominent involvement of IFNG in responses to malaria infection is well established, much less is understood about production of T1IFNs. Studies have shown that malaria extracts can induce IFNA from human pDCs in vitro
, and have documented IFNA induction in P. chabaudi
and P. berghei
-infected mice 
. Using a variety of approaches, we have demonstrated that T1IFNs are indeed produced during in vivo
infection with P. chabaudi
, and that both pDCs and RPMs are the key cellular sources that contribute to the systemic T1IFN pool.
Although the protective role of T1IFNs in viral infections is well established, in some bacterial infections and autoimmune disorders, T1IFNs appear to exacerbate disease 
. Similar to viral infections, our functional studies indicate that T1IFNs act redundantly with IFNG to activate mechanisms that protect against malaria disease. Together, our findings reveal redundancies at several different levels: first, at the level of multiple molecular sensing pathways in RPMs feeding into T1IFN production; second, at the level of multiple leukocyte populations generating systemically available T1IFNs; and finally, at the level of T1IFNs conferring protection that is redundant with IFNG. We suggest that this tiered redundancy is widespread in immunological systems but has been overlooked due to absent or mild phenotypes in organism-level assays.
T1IFNs frequently originate from pDCs, which are also known as “interferon producing cells” due to their ability to produce more T1IFNs than any other cell type in human blood 
. Our observation that pDCs produce IFNA and IFNB during malaria infection is in line with the general function of pDCs and similar findings from Voisine et al.
. However, we have demonstrated that RPMs also contribute significantly to total T1IFN production during the response to P. chabaudi
, indicating that these macrophages play a role in early immune activation during malaria infection. We estimate that roughly 3000 pDCs and 1000 RPMs per spleen produce high levels of T1IFN, and the comparable fluorescence levels of these populations in Ifnb-Yfp
reporter animals suggest that pDC and RPM are capable of transcribing similar levels of Ifnb
. Whether or not this corresponds to similar levels of IFNB production on a per-cell basis remains to be determined; regardless, our findings contribute to the increasing body of literature indicating that macrophages and other non-pDC populations are significant sources of T1IFNs in vivo
It is likely that the localization of the infections at the tissue, cellular, and sub-cellular levels defines in part which leukocytes respond and in what manner. This is likely to be the case for T1IFN production by RPMs in malaria infection: ultrastructural studies have demonstrated that RPMs are capable of phagocytosis of both whole infected erythrocytes and parasites that have been “pitted” from infected erythrocytes in the spleen 
, and trafficking studies using stained infected erythrocytes have demonstrated localization to the splenic red pulp 
. Although these studies only examined splenic organization during the time of peak parasitemia, it was reasonable to expect that RPMs would also function as early detectors of malaria parasites due to their inherent role in filtering parasites from the blood. We have demonstrated that this is indeed the case, despite the low parasite load during early sub-patent infection, and that RPMs respond by producing T1IFNs and a host of additional chemokines and cytokines. To the best of our knowledge, this is the first demonstration of production of an immunomodulatory cytokine by RPMs during early malaria infection.
We have found that TLR9-MYD88-IRF7 signaling is required for full T1IFN expression in RPMs, similar to the role of this pathway in pDC 
. This is at odds with the fact that no in vitro
studies of TLR9 activation have reported IFNA production in mouse pDCs or macrophages, but it is possible that malaria ligands may be less potent than synthetic ligands and therefore require additional activating signals from other leukocyte populations present in vivo
. Obvious candidates for such signals include cytokines that signal through the MAP kinase and NF-kappa B pathways, which participate in Ifnb
induction through the heterodimeric transcription factors ATF-2/c-Jun and p50/RelA 
. Consistent with this possibility, inhibition of NF-kappa B signaling in mice infected with West Nile Virus decreases IFNB production 
. Further studies will be required to understand the relative contributions of these pathways in vitro
and in vivo
, and also to identify the pathway(s) responsible for residual levels of T1IFN production in the absences of TLR9 and MYD88.
T1IFNs can augment their own expression through a feed-forward signaling loop, but for P. chabaudi
, only Ifna,
induction appears to rely on IFNAR1-dependent amplification. This result is similar to observations from Listeria
infection, in which IFNB generation is essentially unaffected by the absence of IFNAR1 whereas IFNA production is severely diminished 
. Similarly, expression of Ifna
by cDCs during West Nile Virus infection was diminished in mice lacking IFNAR1, whereas Ifnb
expression was not 
. Thus, our data extend the paradigm of IFNB being induced prior to amplification loop-dependent production of IFNA, as demonstrated in viral and bacterial systems, to infection with a protozoan parasite. With regard to Ifna
induction by P. chabaudi
, we observed that Ifnar1−/−
mice exhibit similar levels of reduction, consistent with observations from other systems that these molecules are both required for T1IFN amplification 
Although RPMs produce T1IFNs and other cytokines during early infection, mice lacking RPMs clear parasites with kinetics identical to control animals. This result was surprising given the general belief that RPMs contribute to control of parasitemia through phagocytic mechanisms 
. Furthermore, we observed that RPMs act as early sentinels of infection and produce cytokines that ultimately contribute to elimination of infection. Given our observation that pDCs also produce T1IFNs, it is possible that all of the important functions of RPMs are redundant with other leukocyte subsets. For example, splenic monocytes are capable of phagocytosis of P. chabaudi
, and this population undergoes expansion near the time of peak parasitemia in both SpiC+/−
mice (Fig. S7C
). Our data indicate that the Ly6clo
monocytes are also significantly increased in frequency in RPM-deficient mice, suggesting the possibility that this subset could be providing redundancy with RPMs. Although the exact mechanism requires further investigation, our data indicate that the important role of the spleen in clearance of malaria infection is due to functions that are not specific to RPMs.
In summary, our results demonstrate that T1IFNs play a redundant but important protective role during experimental malaria infection. These T1IFNs are derived from both pDCs and RPMs, which are thus identified as the major populations responsible for early innate recognition of malaria infection. Future work will reveal how these innate populations and T1IFNs promote the development of an integrated immune response that can ultimately resolve malaria infection.