Spatiotemporal Dynamics of Immune Cells in Early Left Ventricular Remodeling After Acute Myocardial Infarction in Mice.

Myocardial infarction remains a leading cause of morbidity and death. Insufficient delivery of oxygen to the myocardium sets into play a complicated process of repair that involves the temporal recruitment of different immune cells so as to remove debris and necrotic cells expeditiously and to form effective scar tissue. Clearly defined and overlapping phases have been identified in the process, which transitions from an overall proinflammatory to anti-inflammatory phenotype with time. Variations in the strength of the phases as well as in the co-ordination among them have profound consequences. Too strong of an inflammatory phase can result in left ventricular wall thinning and eventual rupture, whereas too strong of an anti-inflammatory phase can lead to cardiac stiffening, arrhythmias, or ventricular aneurisms. In both cases, heart failure is an intermediate consequence with death being the likely outcome. Here, we summarize the role of key immune cells in the repair process of the heart after left ventricular myocardial infarction, along with the associated cytokines and chemokines. A better understanding of the immune response ought to lead hopefully to improved therapies that exploit the natural repair process for mending the infarcted heart.

Curing mosquitoes with genetic approaches for malaria control.

Malaria remains a persistent global public health challenge because of the limitations of current prevention tools. The use of transgenic mosquitoes incapable of transmitting malaria, in conjunction with existing methods, holds promise for achieving elimination of malaria and preventing its reintroduction. In this context, population modification involves the spread of engineered genetic elements through mosquito populations that render them incapable of malaria transmission. Significant progress has been made in this field over the past decade in revealing promising targets, optimizing genetic tools, and facilitating the transition from the laboratory to successful field deployments, which are subject to regulatory scrutiny. This review summarizes recent advances and ongoing challenges in 'curing' Anopheles vectors of the malaria parasite.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

CLIPB4 is a central node in the protease network that regulates humoral immunity in Anopheles gambiae mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPB4 Is a Central Node in the Protease Network that Regulates Humoral Immunity in Anopheles gambiae Mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

The environment and species affect gut bacteria composition in laboratory co-cultured Anopheles gambiae and Aedes albopictus mosquitoes.

The midgut microbiota of disease vectors plays a critical role in the successful transmission of human pathogens. The environment influences the microbiota composition; however, the relative mosquito-species contribution has not been rigorously disentangled from the environmental contribution to the microbiota structure. Also, the extent to which the microbiota of the adult sugar food source and larval water can predict that of the adult midgut and vice versa is not fully understood. To address these relationships, larvae and adults of Anopheles gambiae and Aedes albopictus were either reared separately or in a co-rearing system, whereby aquatic and adult stages of both species shared the larval water and sugar food source, respectively. Despite being reared under identical conditions, clear intra- and interspecies differences in midgut microbiota-composition were observed across seven cohorts, collected at different time points over a period of eight months. Fitting a linear model separately for each OTU in the mosquito midgut showed that two OTUs significantly differed between the midguts of the two mosquito species. We also show an effect for the sugar food source and larval water on the adult midgut microbiota. Our findings suggest that the mosquito midgut microbiota is highly dynamic and controlled by multiple factors.