Abstract

Malaria control is challenged by parasite drug resistance and inadequate vaccine protection. Although mosquito gut microbes regulate Plasmodium development, integrated analyses of microbiota-Plasmodium-host interactions are lacking. Innate immunity imbalance is the primary driver of disease pathogenesis. This review proposes a unified “microbiota-barrier-innate immunity” axis hypothesis. Under physiological conditions, gut microbiota modulates host innate immunity via metabolites and maintains barrier integrity. However, Plasmodium infection disrupts this axis, causing microbiota imbalance, metabolic derangement, intestinal barrier leakage. The ensuing innate immune dysfunction exacerbates the pathology of malaria. Based on this, we constructed a hierarchical three-tier foundational-targeted-precision intervention pyramid tailored to diverse settings, particularly African regions, with endogenous and technological feasibility. This framework, progressing from foundational nutritional regulation and targeted microbial intervention to precision bioengineering, provides an implementable malaria control strategy for resource-limited African areas, addressing drug resistance and vaccine shortages. It offers a translatable eco-immunological approach to accelerate global malaria elimination.

1 Introduction

Malaria thus constitutes a considerable danger to human existence as a widespread infectious disease, which is endemic to tropical and subtropical regions. The continuance in its control is still beset with a spectral number of structural obstructions, and the disease has not yet been suppressed. Global malaria control still faces a very difficult situation. In 2023, the number of cases moved in the opposite direction of the expected decline and reached 263 million, with about 597,000 deaths; this mortality level was far above international targets. The disease burden is also unevenly distributed, with Africa accounting for 94% of cases and 95% of deaths worldwide (1).Clinically the disease has three forms, namely, severe or complicated, mild or uncomplicated, and asymptomatic (2). The host’s response to infection varies significantly and is influenced by multiple factors. These include parasite protein genetics, co-infections, comorbidities, ethnic background, geographical location, and microbiome composition, as well as the host’s nutritional and genetic background (3, 4). During the progression from infection to the blood stage of the disease, the host’s immune responses are sequentially initiated. This process begins with physical barriers such as the skin, followed by innate immunity mediated by cells like dendritic cells. Ultimately, it develops into adaptive immunity such as the production of cytophilic antibodies (5).

1.1 Bottlenecks in malaria control

The transmission of Plasmodium vivax is highly region-specific. A large-scale genomic study in Brazil supports this. South American parasite populations form a distinct global subgroup. Within Brazil, the parasites are highly differentiated. They form at least seven genetic clusters. The study also identified mutations and selection signals specific to or common in Brazil. These signals are concentrated in two gene groups. One group relates to antimalarial drug sensitivity, such as pvmdr1 and pvdfr-ts. The other group relates to the mosquito transmission stage, such as pvcrmp3, pvp47, and pvp48/45 (6). This finding indicates that the Plasmodium populations in Brazil have a unique evolutionary trajectory. The regional drug resistance-related mutations and local mosquito-borne adaptive evolution collectively highlight the limitations of strategies based on uniformity. Moreover, the effect of climate change on transmission may be contrary to conventional thinking. In regions of Africa and Asia where transmission is intense, when temperature rises above an optimal level the vectorial capacity of Anopheles mosquitoes may be sharply curtailed or completely destroyed (7). This indicates that global warming will not lead to a uniform acceleration of malaria transmission. Instead, due to the heterogeneity of climatic conditions across different subregions within areas of high transmission, its impact on malaria spread is not uniform. It is more likely to reshape geographic distribution patterns with differential effects.

Apart from ecological factors, the emergence and spread of multidrug resistance in Plasmodium parasites to the key drug artemisinin and its combination therapies has led to a steep decline in the cure rates of traditional artemisinin-based Combination Therapy. In response to the known resistance crisis in hotspots such as Cambodia and Thailand, researchers have developed novel approaches like Triple artemisinin-based Combination Therapies (TACTs) to combat resistance. These therapies work together effectively. They work together effectively through multiple mechanisms, including inhibition of nucleic acid synthesis, disruption of heme detoxification, and generation of toxic free radicals. Each drug uses a unique mechanism. Even if the parasite develops resistance to a single drug, it struggles to simultaneously evade the concerted action of three distinct mechanisms. TACTs can increase the cure rate to over 95% and effectively delay the development of resistance. However, these new solutions have also led to secondary bottlenecks. Although TACTs are well-tolerated, they suffer from mild QTc prolongation and increased vomiting. Additionally, the problems with TACT are its high cost, unreliable supply, and ethical issues that hamper their availability and widespread use in limited-resource environments (8), and place the global malaria control project in a vicious cycle between drug resistance and adequate countermeasures.

RTS,S/AS01, recently approved by the WHO for use in African children, represents the current state of the art in this field. The core component is a virus-like particle that expresses the 19 NANP repeats of the circumsporozoite protein of Plasmodium falciparum and the C-terminal region of the 3D7 isolate, fused to hepatitis B surface antigen. In addition, the vaccine contains unmodified HBsAg and the adjuvant AS01E (9). The vaccine was shown to provide about 50% protection for up to 14 months after vaccination in African children in phase 3 trials, but this protection waned with time (10, 11). Also, the IFN-γ response induced by the vaccine mainly comes from NK cells rather than T cells, suggesting that CD8+T cells may not be fully activated. Furthermore, it may also fail to fully mobilize and activate NK cells and other lymphocytes with innate-like characteristics over a long period of time, whereas these cells play a crucial role in controlling early intrahepatic stage infection and establishing a rapid immune response defense (12, 13).

Recent research further challenges the traditional paradigm focused solely on total antibody titers. Protection against malaria shows no correlation with total IgG levels but is significantly associated with specific IgM and IgG subtypes targeting α-galactose (14). Antibodies targeting a new β-ctCSP epitope have also been shown to be present in subjects receiving the RTS,S/AS01 vaccine that cause broad reactivity against different field-isolated strains and produce an inhibition of infection (15). This evidence collectively indicates that future vaccine design strategies must move beyond a single-antigen-epitope approach and instead aim to shape an innate immune landscape based on the specific antibody subtypes. This direction provides a key path for improving the breadth and persistence of existing vaccine protection.

1.2 Focusing on the central role of innate immunity

In the deep mechanism of the bottleneck mentioned above, innate immunity plays a crucial role. It not only serves as the first line of rapid defense, but also plays a crucial role in shaping the pattern of anti-malarial immune responses. The traditional binary view of “pathogen-host” has been insufficient to better interpret the pathology and immune dysregulation of malaria. Recent studies have shown that gut microbiota plays a critical role in this process (16, 17). Due to differences in the abundance of Lactobacillus and Bifidobacterium in mice, genetically identical mice from different sources exhibited significantly divergent parasite loads and mortality rates after Plasmodium infection (18). This phenomenon reminds us that the dynamic tripartite interaction of “host-pathogen-microbiota” should be included in the research core.

At the host level, genetic polymorphism of C3, the main agent of innate immunity, can influence the risk of children developing severe malarial anemia (19). At the transmission stage, the newly uncovered evidence indicates that functionally intact C3 in the host is required for Plasmodium to infect mosquitoes and complete its life cycle successfully. C3 directly lyses the anti-malarial symbiotic bacterium Elizabethkingia anophelis in the mosquito midgut following its activation through the alternative pathway. E. anophelis prevents the development of Plasmodium in the midgut of the mosquito by inhibiting the conversion of the zygotes to ookinetes, and C3’s killing of these bacteria indirectly removes a development block for transmission of the parasite. On the contrary, if host C3 is deficient, or if the activation of C3 is inhibited by the factor B inhibitor LNP023, then the abundance of E. anophelis in the mosquito midgut increases, and transmission of the parasite to mosquitoes is heavily suppressed. Further, aberrant C3 activation rapidly reduces the efficacy of Transmission-Blocking Vaccines such as those providing immunity against Pfs25, but inhibiting C3 activation significantly enhances their transmission-blocking efficacy (20). This interaction between the Plasmodium parasite and the mosquito host not only expands our understanding of the ecological functions of innate immune molecules, but also reveals the unique strategy employed by Plasmodium to utilize the host complement system to facilitate its transmission. This characteristic further underscores the importance of microbiota in regulating C3 function.

Drawing on the preceding portrayal of bottlenecks in malaria control and core principles of the “host-pathogen-microbiota” paradigm, we put forward an integrative theoretical concept of the “microbiota-barrier-innate immunity” axis at the heart of burgeoning malaria pathology. In a healthy state, the gut microbiota secretes metabolites such as Short-chain Fatty Acids (SCFAs), which strengthen intestinal epithelial tight junctions and mucus secretion, enhancing barrier integrity. The microbiota and “barrier” work in concert as a calibration center, fine-tuning the functional states of innate immune cells such as macrophages, NK cells, and Vδ2+ γδ T cells, through key signaling pathways, to enact an effective but restrained system-wide innate immunity. During Plasmodium infection, the parasite first disrupts the microbiota structure, initiating dysbiosis, and second, disrupts the intestinal barrier allowing microbial translocation and inflammation to ensue. This “leakiness” interrupts the calibration signals to the innate immune and complement systems: disturbed signaling, many of the immune effects triggered at the cellular level seem to result in dysfunctional macrophages, dendritic cells, and γδ T cells, driving malaria pathogenesis and transmission. How the microbiota tunes the complement system in homeostatic regulation is thus key to comprehending the physiologic “calibration center” of this axis, aiding it’s seeming collapse entwined with malaria pathology. Guided by this integrated construct, we highlight below specific homeostatic regulatory mechanisms of this axis under healthy conditions, and the Plasmodium-induced disruptive forces that follows, laying theoretical groundwork for subsequent intervention strategies.

1.3 Review focus, conceptual framework, and scope boundaries

This review is designed to lay out, in a systematic way, how mosquito vectors and host gut microbiota participate in malaria transmission, and how these findings can be translated into practical applications. The primary focus is the malaria system transmitted by Anopheles mosquitoes, with particular attention to the adult mosquito midgut as a key ecological niche. This midgut environment is not only where Plasmodium sporogony takes place, but also where tripartite interactions among the microbiota, the parasite, and the host immune system are most directly manifested. At the same time, to maintain an integrated perspective, the human host gut microbiota is also included as part of the discussion, viewed as an important node in systemic immune regulation, thereby supporting a cross-host analytical line that follows “vector–pathogen–human.”

To address these questions in detail, the review uses a multidimensional integrative approach that combines laboratory basic research, animal models, and field epidemiology in Africa. Laboratory work helps clarify fine-scale mechanisms under controlled settings; animal models offer a reproducible way to test causal links; and field studies help assess ecological validity and translational value under real conditions. Each approach also has clear limitations: animal models cannot fully represent human-specific factors such as nutrition, coinfections, and behaviors, and observational field studies usually cannot prove causality. For this reason, when results across evidence sources do not align, they need to be interpreted critically and in an integrated manner.

For theoretical organization, the review follows “microbiota-immunity-parasite” triadic interactions as the central thread. These interactions are discussed across three complementary disciplinary dimensions. From an ecological perspective, attention is given to how community structure influences Plasmodium fitness; from an immunological perspective, the focus is on how the microbiota tunes innate immunity through metabolites and how Plasmodium can exploit these processes; and from a translational perspective, the aim is to gradually turn basic findings into implementable intervention strategies, including nutritional interventions, microbiota regulation, and bioengineering technologies, so that a precision public health system can be developed for resource-limited settings in Africa.

On this basis, the review first outlines the homeostatic regulatory mechanisms of the “microbiota-barrier-innate immunity” axis under healthy conditions; it then examines how Plasmodium infection disturbs this axis, producing dysbiosis, barrier leakage, and immune dysregulation; and finally, using mechanistic insights as a foundation, it proposes a three-tier intervention pyramid oriented to African field settings and discusses practical implementation routes, enabling systems, barriers, and future directions. The overall goal is to provide malaria control with a clear and workable translational path that connects theory with practice and links laboratory research to field deployment.

2 The “microbiota-barrier-innate immunity” axis in health2.1 Gut microbiota homeostasis and the immunometabolic regulatory network

As a complex ecosystem, the structural stability of the gut microbiota is the bedrock on which gastrointestinal homeostasis and host immunity rest. Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria are the four core phyla in the gut of healthy adults (21). The main regulatory mediators is the variety of metabolites synthesized by the microbiota which together comprise the microbiota-immune chemical signaling network. Metabolites can be roughly classified by their origins into three major categories: the first is metabolites generated by fermentation or degradation of dietary fiber; the second is host-derived metabolites that have been modified by microbes; and the third is de novo synthesized microbial metabolites, including effector molecules recognized by the host (22). During malaria infection, these various classes of metabolites might have differential immunomodulatory effects that provide a rationale for a metabolite target database for the precise regulation of the innate immunity.

Homeostasis of gut microbiota is important for preventing aberrant activation of inflammatory pathways such as cGAS-STING. Dysbiosis may lead to immune disorders and aberrations in innate immune pathways. For example, in the STING-related infantile-onset vascular disease mouse model, the decrease of Short-chain Fatty Acid (SCFA) producing bacteria and the rise of segmented filamentous bacteria lead to the increased levels of microbially- and host-derived cyclic dinucleotides, leading to STING pathway activation and consequently exacerbated inflammatory responses (23). In addition, pathogenic DNA from the lethal malaria parasite Plasmodium yoelii Nigeria subspecies N67C clone activated the cGAS-STING signaling pathway in innate immunity. This activation predominantly induced late-stage IL-6 production predominantly through the MyD88-p38 axis and increased CD11b+ Ly6Chi pro-inflammatory monocytes. Both of these suppressed effective adaptive immune responses and increased host mortality. In contrast, cGAS or STING knockout mice exhibited significantly prolonged survival upon infection (24). These findings collectively establish the STING pathway’s core hub connecting malaria infection and pathological inflammation.

2.2 The immunomodulatory effects of short chain fatty acids

SCFAs are well-recognized microbiota-dependent metabolites produced in the gut through the fermentation of dietary fiber, principally including butyrate, propionate and acetate. As key mediators of microbiota-host communication, SCFAs fulfil numerous functions in regulating immune responses, alter intestinal integrity, and calibrate systemic immune homeostasis, principally via HDAC inhibition and GPCR engagement (25).

In the context of immune regulation, SCFAs can influence the functional polarization of innate immune cells. In the lungs, they shape the immune repertoire by activating G protein-coupled receptors including GPR43 (26). Meanwhile, the regulatory effect of SCFAs on macrophage function has also been directly observed in intestinal anti−infection immunity. The gut microbiota metabolite butyrate might strengthen the antibacterial ability of differentiating macrophages through inhibition of the histone deacetylase 3 (HDAC3) enzyme (27). In addition to this, SCFAs play a major role in the orchestration of intestinal circadian rhythms and epithelial homeostasis. Intestinal organoid study uncovered that SCFAs generated by specific gut microbes like Clostridium and Parabacteroides goldsteinii can substantially shift the phase of circadian oscillations in epithelial cells through HDAC inhibition (28), highlighting the direct regulatory capacity of microbial metabolites on host physiological rhythms.

Clinical intervention studies corroborate the pivotal involvement of SCFAs here, with the Mediterranean diet reported to significantly enhance fecal content of propionate and butyrate, accompanied by improved intestinal permeability markers (29). Therefore, in the context of malaria, maintaining sufficient SCFAs levels may be beneficial for modulating innate immune responses and increasing resistance of the host to Plasmodium infection.

2.3 Maintenance of barrier integrity

The healthy intestinal barrier is a multi-layered and highly coordinated defense system that not only provides physical isolation but also has an active immune surveillance function, collectively maintaining host homeostasis. The physical barrier is mainly composed of intestinal epithelial cells and tight junction proteins between them. A mucus layer secreted by goblet cells covers the surface, effectively separating the gut microbiota from the epithelial surface. Furthermore, complete barrier function also includes its superficial immune barrier. In particular, secretory immunoglobulin A secreted by the mucosal immune system plays an irreplaceable role in maintaining microbiota homeostasis and preventing pathogen adhesion (30).

Clinical data collected from patients who underwent splenectomy and patients with variant immunodeficiency diseases provide strong evidence for the role of the immune barrier. The absence of IgM memory B cells leads to a deficiency in the secretion of SIgA in mucosal lymphoid tissues; function of the mucosal barrier is compromised, which allows pathogens to cause more severe symptoms of infection (31). This strengthens the concept that IgM memory B cells are important for mucosal SIgA homeostasis and mucosal barrier integrity. Thus, we can hypothesize that although individuals in malaria-endemic areas have normal immunity, their mucosal B-cell function may be damaged due to malnutrition and/or repeated infections, which might further exacerbate microbial dysbiosis and render them susceptible to malaria.

2.4 Calibration and priming of innate immune cells

The host's innate immune system can respond quickly after sensing pathogen invasion. For example, during Plasmodium yoelii infection, infection activates macrophages and polarizes them towards an M1 phenotype, aiding their phagocytic function. This was observed to correlate with a state of TLR7 upregulation and macrophage STAT3 activation, which the authors report to be mediated by the action of the TLR7-STAT3 pathway. Depleting macrophages via clodronate liposomes, the authors found from their study that the host suffers more severe parasitemia and shows greatly compromised adaptive immune responses (32). Taken together, these results suggest that the TLR7-STAT3 signaling pathway, by influencing the functional activation of macrophages at an early stage of malaria infection, is required for the subsequent initiation of an adaptive immune response. This demonstrates a positive role of this pathway in early host defence against infection.

Beyond affecting myeloid immune cells through similar modalities, microbial signals functionally shape innate-like lymphocytes as well. One of the primary protective mechanisms provided by natural killer (NK) cells in healthy individuals is antibody-dependent cellular cytotoxicity (ADCC), and NK cells activated by appropriate IgG specific to antigens such as PfEMP1 or RIFIN are able to kill infected red blood cells in individuals from endemic areas (33). When NK cells are activated by specific antibodies against Plasmodium parasites, they can degranulate and release IFN-γ; this Ab-NK response has cross-strain protective ability and can enhance inhibition of parasite invasion into red blood cells (34).

Vδ2+ γδ T cells, serving as a critical bridge between innate and adaptive immunity, are functionally capable of controlling blood-stage Plasmodium infection in healthy individuals. The cells selectively recognize phosphoantigen-BTN3A1 complexes displayed on the surface of infected red blood cells (iRBCs) via their T cell receptor (TCR) and through the formation of an immune synapse. They then lyse the iRBCs and destroy intracellular parasites through a direct contact-dependent mechanism involving phosphoantigens and granule granzyme secretion. In parallel, Vδ2+ γδ T cells can phagocytose opsonized iRBCs via a CD16-dependent mechanism, leading to diminished parasite replication and thus facilitating a dual mechanism for protection from malaria (35). In addition, Vδ2+ γδ T cells are important in mediating the sterile immunity produced by vaccination with the PfSPZ vaccine. Furthermore, those individuals with a greatly expanded Vδ2+ γδ T cell population are more likely to benefit from vaccination. In the absence of γδ T cells, the development of CD8α+ dendritic cells in the liver of vaccinated mice is not seen (36). Thus a well-developed Vδ2+ γδ T cell repertoire is a critical determinant of whether the host immune system is able to mount an effective immune response against vaccination.

Upon repeated exposure to malaria, the innate immune system undergoes dynamic reorganization. The initial emergent response mediated by Vδ2+ γδ T cells may no longer be sustainable in quantity and therefore Vδ1+ γδ T cells play a compensatory role. As a result of the increased number, clonal selection and expansion of Vδ1+ γδ T cells, this population differentiates from a naïve state into lytic effector cells which express high levels of perforin and granzyme (37).This shift in emphasis, from Vδ2+ towards Vδ1+ subset, highlights the plasticity and adaptability of innate-like lymphocytes with respect to the establishment of long-lived anti-malarial immunity.

Therefore, under homeostatic situations, a core “microbiota-barrier-innate immunity” axis exists that ensures anti-malarial homeostasis (Figure 1). The molecular circuitry of this axis reveals how symbiotic bacterial metabolites fortify the barrier and tune innate immune cells. The schematic in Figure 1 depicts the regulatory circuitry of this core homeostatic axis, whereby the core microbiota (via metabolites such as SCFAs) fortifies the intestinal epithelial barrier and tunes innate immune macrophages, NK cells and γδ T cells. This serves as an intuitive visual framework for the “microbiota-barrier-innate immunity” axis with key components drawn in.

Homeostatic “core microbiota–barrier–innate immunity” axis in healthy individuals. Arrows (→) indicate induction, promotion, or enhancement; “↑”denotes an increase; “↓”denotes a decrease. IEC, intestinal epithelial cell; GC, goblet cell; Mφ, macrophage; DC, dendritic cell; NK, natural killer cell; γδ T, gamma-delta T cell; SCFAs, short-chain fatty acids; GCM, goblet cell mucins; HDAC, histone deacetylase; PI, Plasmodium infection; iRBC, infected red blood cell.

3 Causal perturbation and remodeling of the “axis” by Plasmodium infection3.1 Evidence from clinical and preclinical studies3.1.1 Human studies: linking dysbiosis and disease

The gut microbiome of malaria patients is characterized by a distinctive state of inflammatory dysbiosis. Central to this phenomenon is not the loss of microbial diversity but rather a pathological remodeling of the community structure. Strong evidence is derived from several cohort studies in Mali. A metabolomic analysis by Schmidt et al. (38), using children susceptible to malaria as the test population, showed significant elevations in fecal inflammatory markers such as deoxynosine and long-chain fatty acids, along with concurrent elevation of markers of repair such as pseudouridine and hypoxanthine.

Finding from Kodio et al. further confirmed that the gut bacterial community structure is significantly correlated with clinical malaria episodes and asymptomatic Plasmodium falciparum infection, with the counterintuitive observation that the higher the bacterial OTU richness, the higher the risk for such malaria episodes. During follow-up, 25 bacterial biomarkers related to malaria susceptibility or resistance were identified, including 17 susceptibility-associated taxa and 8 resistance-associated taxa (39). Most susceptibility-associated taxa belonged to pro-inflammatory genera; for instance, hypermucoviscous multidrug-resistant Klebsiella pneumoniae ST25 can infect intestinal epithelial cells, lower cell viability, disrupt tight junctions, and upregulate the expression of multiple inflammatory factors (40). Clostridium perfringens beta2 toxin can trigger apoptosis in porcine intestinal epithelial cells, increase the expression of several interleukins, and weaken intestinal barrier function (41). In addition, early exposure to Enterobacteriaceae may shape the inflammatory state of the gastrointestinal tract and may modify inflammatory patterns and responsiveness to pathogens (42). In contrast, resistance-associated taxa were mainly beneficial short-chain fatty acid–producing bacteria, such as Bifidobacterium and Lactobacillus (43). Taken together, these findings imply that the high OTU richness observed during malaria infection may represent community-structure dysregulation rather than a stable healthy state.

Although current studies have described characteristic gut microbiota dysbiosis patterns linked with malaria infection, a key limitation is that clear causal relationships cannot be firmly established. The apparently paradoxical pattern of “high OTU richness accompanying high risk” suggests that the community state being measured is more likely an infection-driven inflammatory ecological remodeling, instead of a pre-existing driver of host susceptibility. Moreover, much of the literature still remains focused on species-level association testing, while possible functional mechanisms remain underexplored. Future research needs to integrate longitudinal causal experiments with functional metabolomics to define the roles of key microbial functional pathways in this context. Accordingly, microbiota-based approaches for malaria intervention should not rely on simplistic strategies to increase diversity; they need targeted therapies that can precisely reshape microbial functional ecological niches.

Current research has largely focused on specific regions in Africa, and the general applicability of the “microbiota-barrier-innate immunity” axis requires further validation across populations with varying malaria transmission intensities and ethnic backgrounds. For example, among Hmong and Karen individuals who migrated from Southeast Asia to the United States, a significant decline in gut microbiome diversity has been observed after immigration. American-associated bacterial strains and functional profiles gradually replaced native microbial communities, and this shift has been linked to an increased risk of metabolic diseases (44). The westernization of a living environment and food patterns can create a corresponding westernized gut microbiome that may, in turn, alter the host’s immune responses to multiple diseases, including malaria.

Studies from Odisha, India, report that infection with soil-transmitted helminths negatively correlates with certain gut bacterial taxa including Lactobacillus and Lachnospira. In addition, the relative abundance of Lactobacillus was more dependent on soil-transmitted helminth infection status than on Plasmodium infection status, and vice versa (45). This indicates that in the context of polyparasitism the gut microbiome could regulate susceptibility to a range of pathogens via shared immunoregulatory pathways. These findings further suggest the existence of a conserved “microbiota–barrier–innate immunity” axis that may operate across geographical locations and parasite species.

The aforementioned research conclusions are strongly influenced by geographic context. The specific relationship between microbiota OTU richness and malaria risk reported in the Malian cohort may not be reproduced in other high-transmission areas such as Southeast Asia or South America. Such heterogeneity may reflect differences in human genetic background, dominant Plasmodium strains, local patterns of endemic coinfections, and dietary structure. In addition, an earlier immigration study reported that after moving from Southeast Asia to the United States, the gut microbiota of Hmong and Karen groups shifted toward Westernization, which may alter immune-response profiles. Therefore, a region-specific “microbiota–disease” association pattern should not be directly extended to all malaria-endemic regions. Mechanistic models and intervention planning need to explicitly account for this geographic and population-level heterogeneity and support regionally customized research paradigms.

Extreme clinical phenotypes are important supporting evidence of the above causal chain. A case of a patient in the second trimester of pregnancy with sickle cell disease illustrates that when Plasmodium falciparum parasitemia is complicated by autoimmune hemolytic anemia, hemoglobin concentration drops sharply, and lactate dehydrogenase and bilirubin levels increase significantly. After the administration of antimalarial drugs, the parasites were eliminated and the hemolytic symptoms of the patient were eliminated (46). The pre-existing dysbiosis of gut microbiota and low-grade inflammation of a SCD patient were rapidly aggravated by the dual assault of pregnancy and malaria infection. The antimalarial treatment breaking this vicious circle is an indirect confirmation that the malaria parasite acted as one component of this dual assault that initiated the pathological processes.

3.1.2 Animal models: causal influence of microbiota on disease outcome

The microbial community of the gut is thus itself an independent risk factor determining the outcome of infections, with transplantation experiments in germ-free mice providing direct evidence. Recipients of cecal contents from malaria-resistant mice develop parasitic infections with low burdens while recipients of microbiota from susceptible mice demonstrate high blood parasitemia (18), indicating that certain specific gut communities possess causal ability to suppress malaria.

The gut microbiota can directly prime an innate immune defense pathway independent of adaptive immunity to suppress malaria through a mechanism involving α-galactose-containing polysaccharides on the surfaces of Plasmodium falciparum sporozoites. The level of natural α-gal-specific IgM antibodies is significantly associated with the risk of malaria; higher levels are found in infection-naïve individuals compared to infected individuals, and in older individuals who are less likely to become infected. Mice colonized with Escherichia coli O86:B7 had increased anti-α-gal IgM antibodies and increased resistance to malaria. Furthermore, immunization of α-gal-deficient mice with α-galactose generated high-titer antibodies that mediated complement-dependent cytotoxicity against sporozoites immediately after mosquito bite (47).

From this, we might argue that prior to the activation of the adaptive immune responses, the gut microbiota establishes an intestinal immunological “set-point” for the host, effectively regulating natural antibodies produced by innate-like B cells and conveying susceptibility (or not) to malaria infection. This study identifies the gut microbiota as a key regulatory node of malaria severity and provides a rationale for exploring probiotic intervention and manipulation of the innate immune microenvironment.

Nonhuman primate models provide additional validation. In the longitudinal sampling of the Plasmodium cynomolgi-infected rhesus monkey model, a lower gut microbiota alpha-diversity at peak parasitemia and a significant increase in the relative abundance of Proteobacteria were observed. However, the abundances of Lactobacillaceae, Prevotellaceae, and Spirochaetes were decreased compared to baseline levels. But in the first recurrence stage compensatory enrichment of Lactobacillaceae was demonstrated. At the metabolomic level, significant changes in the tryptophan-kynurenine pathway, and significantly increased expression of L-tryptophan biosynthesis genes have been reported (48). This indicates that the rebounding Lactobacillaceae during recurrence can be recognized as a signal of the host’s self-limiting repair. This discovery links gut microbiota disruption to systemic innate immune inflammation. It is noteworthy that the observed compensatory enrichment of Lactobacillaceae during the first recurrence stage of the disease may be a beneficial innate immune regulatory signal initiated by the host to inhibit excessive inflammation and promote barrier repair.

In summary, clinical observations, animal transplantation experiments, and primate model studies have revealed the role of gut microbiota in malaria infection. They provide evidence from different perspectives. These include correlation, causality, and dynamic hypotheses. However, the levels and strength of these proofs differ significantly. To clearly compare these differences, main research evidence is summarized by its strength (Table 1).

Study type/modelObservationCausal evidenceProposed mechanismLimitationsreferencesHuman Clinical Studies1. The intestinal microbiota of malaria patients presents inflammatory dysbiosis.
2. The bacterial OTU richness is positively correlated with the clinical risk of malaria.
3. Twenty-five bacterial biomarkers associated with malaria susceptibility/resistance have been identified.Direct causal experiments are lacking. Existing studies adopt a correlational design.The phenomenon of higher OTU richness associated with higher risk may indicate a microbiota dysbiosis state driven by the infection itself.1. Core limitation: The correlational study design fails to establish a causal relationship.
2. Most studies only focus on species-level associations, with insufficient exploration of functional mechanisms.(38, 39)Animal Model (Mouse)Mice with distinct indigenous gut bacteria exhibit different levels of parasitemia and mortality following Plasmodium infection.1. Fecal microbiota transplantation (FMT) experiments directly demonstrate that the gut microbiota can causally modulate the outcome of Plasmodium infection.
2. Colonization with a specific strain of E. coli O86:B7 leads to elevated anti-α-gal antibodies and confers resistance to Plasmodium sporozoites.The gut microbiota may prime the host’s early anti-malarial immune status by training innate defense pathways independent of adaptive immunity and regulating the production of natural anti-α-galactose IgM antibodies by innate-like lymphoid cells.Findings from mouse models have not been mechanistically validated in humans. The human immune microenvironment and microbiota composition are far more complex.(18, 47)Animal Model (Non-human Primate)1. The α-diversity of the intestinal microbiota decreases and the relative abundance of Proteobacteria increases at the peak of infection.
2. A compensatory enrichment of Lactobacillaceae is observed during the first relapse phase of the disease.Longitudinal observations reveal the dynamic changes of the gut microbiota throughout the infection course, while no interventional experiments have been performed to verify its functional role.The enrichment of Lactobacillaceae during the relapse phase may represent a host-initiated self-limiting repair signal.The functional role of specific microbial taxa remains at the correlational and hypothetical stage.(48)

Summary of the evidence hierarchy for the association between malaria infection and gut microbiota.

3.1.3 Discrepancies between human evidence and animal models

In the Malian cohort, the result that “the higher the bacterial OTU richness, the greater the risk of malaria episodes” stands in direct conflict with FMT findings in germ-free mice, where “a high-diversity microbiota reduces parasite burden.” This contrast indicates that diversity metrics alone may not have a consistent predictive meaning across different contexts.

Notably, just as the gut microbiome is recognized as a key mediator between the environment and human physiology, disease susceptibility, and drug responses, the genetic background and microbiome of laboratory mice are also important experimental variables that influence research outcomes. Controlling and standardizing the gut microbiome in lab mice can significantly improve the predictive power and applicability of research data when translating findings to human situations (49). This reminds us that when understanding and reconciling differences between studies, the microbiome must be considered as a core experimental variable.

Laboratory studies often start from germ-free or antibiotic-treated mice, which are essentially simplified systems without complex microbial communities. By comparison, the microbiota in field populations reflects an adaptive steady state shaped by many long-term pressures, including repeated malaria exposure, malnutrition, recurrent infections, and broad-spectrum antibiotic use. Under such conditions, increased species OTU richness could be a compensatory response to persistent inflammatory stimulation, and it may even mark the ecological expansion of pathogenic bacteria within a dysbiotic community. At present, there is still no functional evaluation framework that can reliably separate healthy high diversity from pathological high diversity, so the same diversity indicator can receive opposite health interpretations depending on the study setting.

Therefore, in field settings where infection and malnutrition coexist, the microbiota’s functional composition and its ecological relationships are more informative than simple OTU richness counts. Field interventions should not aim to raise diversity in a non-specific way; instead, they should try to restore functional balance through deep sequencing together with metabolic functional analyses.

3.2 Three major pathways of axis disruption by Plasmodium3.2.1 Dysregulation of innate immune cell function3.2.1.1 Macrophages: specific functional subversion

In malaria, the functional disorders of macrophages are directly related to determined pathologies. In the case of severe malarial anemia, macrophages that continually phagocytose the parasites present decreased expression of CD169 on the surface in consequence of losing their anchorage, hence supporting the immature red blood cells (50). Thus a vicious circle of insufficient production of red blood cells and greater destruction is established. In the case of placental malaria, although the number of macrophages and the M1/M2 ratio remain unaltered, the expression of key functional genes such as STAT-6 and ANG-1 is lessened in M2 macrophages. This disrupts the immune homeostasis of pregnancy, with consequent adverse effects on maternal health and fetal development (51). In this connection there appears to be evidence that a deficiency or more likely an imbalance in the microbial metabolites SCFAs may lead to the functional disorders in these macrophages with the consequence of exacerbating malaria pathology.

The function of the macrophage is also precisely regulated through many upstream signaling pathways, implicated in the microbiota–immune axis. The unbalanced M1/M2 ratio of macrophages can be normalized by blocking the paracrine effect of IL-6 (52). Artemisia annua acid can bind to GPR37 on the surface of macrophages, promoting their phagocytic function and enhancing their ability to clear pathogens (53). Gene variants of CSF2 affect granulocyte macrophage colony-stimulating factor expression with downstream consequences for macrophage function that ultimately influence host susceptibility to malaria and severe malarial anemia (SMA) (54). Together, these pathways constitute a functional regulatory network of macrophages, providing multiple potential targets for targeted intervention.

3.2.1.2 Dendritic cells: induction of programmatic paralysis

Infection with malaria can lead to functional suppression of dendritic cells. Infected persons show a decreased frequency and an increased apoptosis of circulating CD1c+ myeloid dendritic cells, with surviving cells being profoundly functionally suppressed. These cells, however, show an increased capacity for tumor necrosis factor secretion, but downregulation of HLA-DR and the co-stimulatory molecule CD86 results in an impaired capacity for antigen presentation. This selective dysregulation of cytokine secretion further adds to the imbalance in immune response polarization of this disease, and makes the establishment of a targeted anti-malarial immune landscape exceedingly difficult (55).

This condition may reflect a programmed form of functional suppression that is triggered by signals from the local microenvironment. For instance, systemic inflammatory response syndrome (SIRS), whether caused by pathogen mimicry or malaria itself, can induce functional paralysis not only in conventional dendritic cells but also across successive generations of these cells. The paralyzed conventional dendritic cells have a different transcriptional and phenotypical profile which is characterized by decreased antigen capture and impaired antigen presentation and an abnormal cytokine production that prevents the proper mediation of anti-malarial immunity. Application of vaccinations like monoclonal antibodies that target one of the cDC receptors or Transforming Growth Factor-beta (TGF-β) antagonism can exhibit a partial restoration of this paralysis and associated immunosuppression (56), indicating that in both cases there is a restoration of the degree of the anti-malarial immunoprecipitation and that there is, a decrease parasitic load in the mice, proving that the programmed paralysis of the dendritic cells is reversible, thus providing an experimental basis for working towards future mechanisms based on a function restoration of dendritic cells.

3.2.1.3 NK and γδ T cells: exhaustion and remodeling

Within the context of chronic infection, NK cells exhibit decreased natural cytotoxic function but an intact ability to mediate antibody-dependent cellular cytotoxicity (ADCC) (57). Areas endemic for malaria and Epstein-Barr virus (EBV) also show an increase in the frequency of functionally exhausted CD56negCD16pos NK cells in children with endemic Burkitt lymphoma (eBL) and a corresponding decrease in highly cytotoxic CD56dimCD16pos NK cells, resulting in an overall loss of cytotoxicity. Additionally, in long-term survivors of eBL, this dysfunctional NK cell phenotype can gradually normalize, suggesting the exhausted state may be reversible (58).

In high transmission environments, as children get older and have reinfections, the expansion capacity and absolute numbers of Vδ2+ γδ T cells are decreased. Submicroscopic parasitemia and immunoregulatory markers such as Tim-3 and CD57 are associated with a reduced capacity for production of the proinflammatory cytokines (59). Repeated infections also lead to a reduction in the percent and response and proliferative capacity of the Vδ2+ γδ T cells, which is associated with a reduced capacity for formation of proinflammatory cytokines. This functional exhaustion is correlated with diminished symptoms during subsequent infections, suggesting that it represents a possible disease tolerance mechanism (60). While this functional exhaustion comes at a cost to the host in acquiring clinical immunity, it also produces a therapeutic window for possibly restoring γδ T cell function by microbiota-directed interventions.

3.2.2 Induction of intestinal barrier leakage3.2.2.1 Regulation by immune cells

IL-4 and IL-13 derived from eosinophils do not participate in eosinophil dependent parasitic transmission regulation, but these cytokines coordinate the protection of intestinal barrier integrity after Plasmodium yoelii infection. Specifically, IL-4/IL-13, which is dependent on eosinophils, controls mast cell activation and prevents intestinal barrier damage and bacteremia caused by infection by regulating eosinophils, macrophages, and Th17 mediated inflammation (61).

The function of the mast cell protease, Mcpt4, is more complicated. After infectious challenge, Mcpt4-deficient mice exhibited elevated intestinal TNF-α and IL-12p40 levels, indicating a more vigorous type I immune response and reduced parasitemia. However, it was also shown that these animals exhibited increased intestinal permeability and disruption of adhesion junctions including E-cadherin (62). It appears that Mcpt4 skews the immune response toward a type II immune response because of the degradation of pro-inflammatory cytokines. Therefore, it reduces the overall immune intensity systemically while indirectly helping to maintain the epithelial integrity of the intestinal barrier.

Mast cell IL-10 is also important in GI barrier protection and this has a sexually dimorphic aspect. Early in infection, mast