Crosstalk between hypoxic cellular micro-environment and the immune system: a potential therapeutic target for infectious diseases.

Mammalian cells generally adapt to hypoxia (low oxygen tension) by activating a major heterodimeric transcription factor known as hypoxia inducible factor (HIF)-1 ( 1 ). HIF-1 is composed of the two main subunits; HIF-1α and HIF-1β. HIF-1α levels are modulated by changes in oxygen partial pressure (pO 2 ) within the cellular micro-environment. On the other hand, HIF-1β are constitutively expressed regardless of changes in the extracellular flux ( 2 ). HIF-1 regulates the robust expression of hypoxia-inducible genes (HIGs) downstream which are mainly involved in glycolysis (mediated by hexokinase), angiogenesis (mediated by vascular endothelial growth factor), erythropoiesis (mediated by erythropoietin), and cellular proliferation (mediated by adrenomedullin) ( 3 ). Mechanistically, HIF-1 activation requires the enzymatic inhibition of the prolyl hydroxylase domain-containing proteins (PHDs), a process which occurs strictly during hypoxia. In a normoxic (normal/optimum oxygen tension) situation, PHDs-mediated hydroxylation of the prolyl residues of the HIF-1α subunit ensures its binding to the von-Hippel-Lindau (VHL) protein, the recognition component of an E3 ubiquitin-protein ligase, leading to an eventual proteasomal degradation ( 2 ). However, hypoxia creates an inhibitory effect on PHD-hydroxylation thereby mediating the stabilization of HIF-1α, subsequent binding of the HIF-1 heterodimer to the promoter regions of HIGs resulting in the induction of those genes ( 4 ) ( Figure 1A ). Accumulating evidence from literature strongly suggest that HIF-1α plays a key role in the immune response to infection and inflammatory process in mammalian hosts ( 5 – 7 ). Essentially, HIF-1α is critical for the bactericidal activities of phagocytes like macrophages and neutrophils, and vital for inhibiting the systemic dissemination of certain bacteria ( 8 ). The role of nuclear factor kappa B (NF-kB) signaling pathway in infectious disease development and control has been well characterized ( 9 , 10 ). Here, we discussed the interaction between HIF-1α and NF-kB, and their crosstalk within vital components of the innate immune system, highlighting the importance of this crosstalk as a plausible therapeutic target for infectious diseases. Schematic representation of the molecular crosstalk between the HIF-1a and NF-kB pathways. During normoxia (normal/optimum oxygen tension), prolyl hydroxylase domain (PHDs)-mediated hydroxylation of the prolyl residues of the HIF-1a subunit enables its binding to the von-Hippei-Lindau (VHL) protein, the recognition component of an E3 ubiquitin-protein ligase, resulting in proteasomal degradation. However, during hypoxia, HIF-1α activation requires the enzymatic inhibition of the PHDs-containing proteins. Hypoxia creates an inhibitory effect on PHD-hydroxylation, mediating the stabilization of HIF-1α, subsequent binding of the HIF-1 heterodimer to the promoter regions of HIGs resulting in the hypoxia inducible genes (A) . The stabilization of HIF-1α stimulates NF-kB activation by negatively modulating the catalytic activity of the inhibitor of nuclear factor Kappa-b (IKKb). NF-kB has several sub-units like P50, P65 and c-Rel which can be activated by many stimuli, including bacterial lipopolysaccharide (LPS), viral pathogens, cytokines or growth factors, leading to the activation of Toll like receptors (TLR) (A) . HIF-1α stabilization enhances the expression of NF-kB-regulated inflammatory cytokines from immune cells like macrophages, neutrophils, T and B cells, after lipopolysaccharides (LPS) stimulation, mediating NF-kB activation of those cells. As shown here, most bone marrow-derived phagocytic cells, essentially granulocytes like neutrophils, monocytes (macrophages in tissues), during peripheral circulation of oxygenated blood in blood vessels, sustain their bactericidal and pro-inflammatory abilities in a "naive state". However, following extravasation and upon invading an infected tissues, they encounter a robust decline in oxygen gradients, and a consequent bolstered activation of their bactericidal and pro-inflammatory abilities ("on-state") (B) . Consequently, HIF-1α stabilization could either inhibit the infection and replication of some pathogens while upregulating others. Created with BioRender.com .

Macrophages are very important hematopoietic cells derived from circulating monocytes upon migration into tissues and becoming differentiated. These myeloid cells play a vital role both in innate and adaptive immune response ( 20 ). They initiate inflammatory responses, phagocytose, and destroy pathogens, and ensure lymphocytes recruitment. They also have the intrinsic ability to show extensive plasticity by undergoing two different types of activation: the classical M1 profile induced by Toll-like receptor (TLR) ligands, inducing a characteristic proinflammatory activity; and the alternative M2 profile, induced by specific interleukins like interleukin (IL)-4 and IL-13), which are largely involved in anti-inflammatory activities ( 21 , 22 ). During an infection or inflammatory process, a typical host response is monocytosis; infiltration and accumulation of these myeloid cells into the infection and inflammatory site ( 23 ). Hypoxia represents an important factor modulating monocytopoiesis, specifically, the maturation from monocytes to macrophages ( 24 ). HIF-1 α and TLRs bidirectionally interact on a plethora of biologically relevant levels ( 5 ). While HIF-1 α regulates the surface expression of various TLRs (e.g., TLR-2, -4, -6, and -9), intracellular signal transduction of several TLRs as aforelisted is often mediated by HIF-1 α . NF-kB is a major downstream player of this pathway ( 5 ). NF-kB remains a major control factor of the mammalian immune system and has been exhaustively reported as being strongly induced by LPS ( 25 ). Interestingly, LPS-induced HIF-1α activation is dependent on NF-kB in both human and murine monocytes ( 26 , 27 ). Functionally, it’s been shown that in response to hypoxia, monocytes switch to a glycolytic metabolism thereby blocking their migration mediated by the chemotactic gradient ( 28 ). Gene expression modulations, such as the upregulation of essential molecules needed for macrophage survival, such as glucose transporter 1 (GLUT1) and their association with hypoxia has been well studied as well ( 29 ). Conversely, in both human and murine macrophages, hypoxia can initiate gene expression in a HIF-independent manner ( 14 ), however, not only through the upregulation of NF-kB, but also activation of transcription factor 4 (ATF4), and early growth response-1 (Egr-1) ( 30 ). The strict importance of HIF-1 α for TLR activation could provide a plausible molecular explanation for the functional importance of HIF-1 α in many pathogens’ defense. The interaction between NF-kB and neutrophils in a hypoxic cellular milieu is critical in influencing immunological responses and inflammatory processes ( 5 ). NF-κB regulates immune-related gene expression, and neutrophils, as major actors in innate immunity, are known for their quick reaction to infections and tissue injury ( 31 ). Neutrophils have a short lifespan and are highly motile cells, largely depending on glycolysis as their major energy source ( 32 ). As they migrate from the circulation to sites of inflammation, they are required to adapt to and function within lower oxygen tensions because the physiological oxygen gradient is often greatly exaggerated in disease situations. Hypoxia bolsters the association of HIF-1α with the p65 component of NF-kB in neutrophils, hence increasing NF-κB-dependent gene expression ( 33 ). Furthermore, the generation of reactive oxygen species (ROS) influences NF-κB activation in neutrophils under hypoxia ( 34 ). Hypoxia causes neutrophils to produce more ROS, which then activates NF-κB ( 34 ). A study on cancer cells revealed a positive feedback loop between ROS and NF-kB, demonstrating that hypoxia-induced ROS generation activates NF-κB in neutrophils, resulting in the overexpression of pro-inflammatory genes ( 35 ). Notably, the connection between NF-κB and neutrophils in a hypoxic milieu is bidirectional since NF-κB activation can impact neutrophil activities in a reciprocal manner ( 35 ). Neutrophil migration, survival, and cytokine production have all been demonstrated to be influenced by NF-kB signaling ( 36 ). Also, NF-κB activation in neutrophils under hypoxia improves survival by upregulating anti-apoptotic genes ( 37 ). HIF-1α-induced activation of NF-κB in neutrophils can also influence the production of cytokines and chemokines, which are important mediators of inflammatory reactions ( 35 ). Dendritic cells (DCs) are vital antigen presenting cells (APCs) between the innate and adaptive immune system ( 38 ). Hypoxia and inflammation contribute significantly towards DC differentiation, migration, and survival, essentially mediated by HIF-1α ( 38 ). An important activity of DCs is migration to inflammatory sites, often characterized by low oxygen levels. Under this condition, there is an upregulation of HIF-1α levels, and immature myeloid DCs may undergo cell death due to hypoxia ( 39 ). However, DC maturation induced by LPS provides protection against hypoxia-induced cell death. This protective activity is mediated by the P13K/AKT pathway, a key intracellular signaling pathway involved in cell growth, motility, survival metabolism, and angiogenesis ( 39 ). Importantly, LPS stimulation in a hypoxic environment leads to higher levels of IL-6 and TNF compared to levels produced in normoxia ( 40 ). For the induction of TNF in hypoxic conditions, there is a need for HIF-dependent upregulation of MAP3K8 (MAPK kinase kinase 8), an upstream player in the p38/MAPK pathway ( 39 ). Conversely, murine experiments with HIF-1α-deficient DCs have shown a decrease in the expressions of IL-12p70, IL-10, TNF, IL-1b, and IL-23 following LPS stimulation in hypoxic bone marrow-derived DCs, strongly suggesting that the induction of these cytokines during hypoxia is independent of HIF-1α in mice ( 38 ). HIF-1α has been shown to be vital for DC migration, and the activation of the chemokine receptor CCR7, resulting in an increased glycolysis in humans ( 39 ). The elevated glycolysis is required for the rapid, yet transient, migration of DCs to draining lymph nodes, needed for the initiation of protective immune responses and the maintaining homeostasis. T cells are central players in adaptive immunity ( 41 ). In T cell development, NF-kB is a vital factor in the early antigen-independent phase of thymocyte differentiation and the final antigen-dependent lineage commitment and post-selection maturation ( 42 ). HIF-1α stabilization in T cells has been shown to occur in response to hypoxia and is essential for maintaining T cell homeostasis and functionality ( 43 ). Moreover, HIF-1α stabilization in T cells has been reported to bolster their survival and proliferation, as well as promote the production of effector cytokines, such as interferon-gamma (IFN-γ) and interleukin-17 (IL-17) ( 43 ). Furthermore, the interaction between HIFs and T cells in a hypoxic microenvironment influences T cell differentiation ( 44 ). HIF-1α has also been implicated in regulating T cell lineage commitment and polarization ( 44 ). Specifically, HIF-1α has been shown to promote the differentiation of T helper 17 (Th17) cells, a subset of CD4+ T cells that play a critical role in inflammatory responses and autoimmune diseases ( 45 ). The study by Dang et al. demonstrated that HIF-1α directly interacts with the transcription factor RORγt, a master regulator of Th17 cell differentiation, thereby promoting Th17 cell differentiation under hypoxic conditions ( 46 ). Moreover, HIF-1α and NF-kB molecular crosstalk can also influence regulatory T cell (Treg) function in a hypoxic microenvironment ( 47 ). Tregs are crucial for maintaining immune tolerance and suppressing excessive immune responses. HIF-1α has been found to modulate Treg stability and suppressive capacity ( 47 ). This crosstalk promotes the stability and immunosuppressive function of Tregs by upregulating the expression of Foxp3, a key transcription factor involved in Treg development and function ( 48 ). Considering the numerous shared activators, inhibitors, and molecular targets between HIF-1α and NF-kB, a deeper insight into their crosstalk in T-cell development and function would greatly advance efforts toward discovering novel targets for T-cell-mediated therapeutic development. B cells are essential components of the adaptive immune system responsible for antibody-mediated immune responses ( 49 ). Currently, there is a dearth of information on the effect of the HIF-NF-kB crosstalk in B cells. However, in a hypoxic cellular microenvironment, the interaction between HIF-1 and B cells significantly modulates B cell development, activation, and antibody production ( 13 ). Understanding the interplay between HIF-1 and NF-kB in B cells in a hypoxic setting could provide valuable insights into the molecular mechanisms governing B cell responses and immune regulation. An interesting aspect of the interaction between HIF-1 and B cells in a hypoxic microenvironment is the effect of HIF-1 on B cell development and differentiation ( 12 ). Hypoxia has been implicated in regulating immunoglobulin production ( 50 ). Specifically, HIF-1α has been shown to promote the generation of B cell precursors and enhance their maturation into antibody-secreting plasma cells ( 50 ). Also, the molecular interaction between HIF-1α and B cells has been connected to immunoglobulin class switching ( 51 ). Moreover, HIF-1α has been shown to regulate the expression of key molecules involved in B cell activation, such as CD40 and CD86 ( 52 ). The physiological process of inflammation is intricate and involves the activation of multiple coordinated signaling pathways in response to stress ( 53 , 54 ). Typically, the response entails the expression of small peptides (e.g., cytokines), glycoproteins (e.g., the cluster of differentiation (CD)], and transcription factors, including NF-κB, which contribute to both anti-inflammatory and pro-inflammatory mediators. NF-κB is recognized as the primary pro-inflammatory family of transcription factors and has been identified as a direct modulator of HIF expression in the context of inflammation and hypoxia ( 53 , 54 ). Generally, chronic inflammatory diseases are characterized by a significant interplay between HIF and NF-κB signaling pathways ( 53 ). Hypoxia-induced activation of HIF-dependent genes, along with impaired hydroxylase activity, contribute to the activation of HIF. Inflammatory ligands activate NF-κB activity, which leads to the expression of inflammatory and anti-apoptotic genes ( 53 ). For example, mice that overexpress HIF-1α in keratinocytes show enhanced NF-κB activity and elevated expression of pro-inflammatory genes resulting in heightened sensitivity to inflammatory stimuli ( 55 ). In contrast, mice lacking HIF-1α in neutrophils revealed the involvement of HIF-dependent NF-κB signaling in the regulation of neutrophil survival. The presence of common target genes and the physical interactions between HIF subunits and NF-κB remains the major reason contributing to their crosstalk in inflammatory settings ( 4 ). Studies on several inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disease, colorectal cancer, asthma, and many others, show the presence of potential points and the similarity in the cellular microenvironment in those conditions, which underscore the importance of the crosstalk between HIF and NF-κB ( 55 – 57 ). There is growing evidence that the crosstalk between these two pathways may be critical in the pathogenesis of a range of infectious diseases, including viral, bacterial, and parasitic infections ( 58 ). Figure 1B provides a short list of viral, bacterial and parasitic infectious diseases that are impacted by hypoxia and consequently could be modulated, either inhibition or upregulation, by the molecular crosstalk between HIFs and NF-kB. Essentially, as described above, the hypoxic response is crucial for the optimum activities of tissue macrophages and infiltrating neutrophils that encounter low oxygen pressure in infected tissues. Also, HIF-1α has been proposed to bolster the expression of inflammatory cytokines that are known to be regulated by NF-κB, particularly in LPS-stimulated macrophages ( 59 ). It has been suggested that the activation of HIF-1α during Infection is particularly attributed to common mechanisms associated with the Infection, such as a hypoxic environment caused by bacterial oxygen consumption ( 59 ). The role of hypoxia on infectious agents is bidirectional. Several studies have shown the activation of HIF-1α in response to infections caused by many pathogens either upregulate or downregulates their replication in host cells ( 60 , 61 ). On the other hand, it has been suggested that the presence of bacteria can also stabilize HIF-1α in both immune cells and epithelial cells ( 60 ). Upon stabilization and activation, HIF-1α initiates the release of anti-microbial peptides like cathelicidins and granule proteases, which facilitate the production of TNF- α and nitric oxide ( 62 ). Considering a highly debilitating infectious disease, tuberculosis caused by Mycobacterium tuberculosis (Mtb), NF-κB is involved in the transcriptional activation of HIF-1 ( 61 ). Studies have observed a higher expression of HIF-1α and related immune responses in Mtb infection both in vivo and in vitro ( 63 , 64 ). When murine macrophages are infected with Mtb, inducible NO synthase (NOS2/iNOS) increases nitric oxide (NO), leading to the inhibition of PHDs and creating a positive feedback loop that results in sustained high levels of HIF-1α and increased macrophage activation ( 61 ). Through the regulation of HIF-1α activation in macrophages during microbial infections, which can result in decreased oxygen levels, NF-κB has the ability to boost glycolytic energy metabolism, the production of angiogenic factors, and the expression of pro-inflammatory cytokines, chemokines, and anti-microbial peptides. As a result, the ability of NF-κB to enhance the expression of HIF-1α broadens its regulatory capabilities, leading to the more effective execution of the host-defense response. Furthermore, modulations in oxygen tension and attendant HIF-1α signaling may also play a vital role in viral tropism and pathogenesis ( 65 , 66 ). Therefore, pharmaceutical agents that modulate the HIFs pathway could provide novel treatment options for infections and associated pathological conditions. However, advanced investigations are required to decipher the complex interactions between different pathogen-specific genes and the crosstalk between HIFs and NF-kB. One aspect of advanced investigations involves unraveling the intricate interplay between HIFs and pathogen-specific genes. Different pathogens have evolved diverse mechanisms to interact with host cells and manipulate cellular processes. Understanding how HIFs modulate the expression of pathogen-specific genes is crucial for deciphering the underlying molecular mechanisms of pathogenesis and identifying potential therapeutic targets. This requires comprehensive studies using advanced genomics, and transcriptomics approaches to characterize the transcriptional changes induced by HIFs during Infection ( 66 , 67 ). Furthermore, investigating the crosstalk between HIFs and NF-kB is of paramount importance. Both HIFs and NF-kB are central players in inflammation and immune responses, and their interactions can significantly influence the outcome of infectious diseases. Deciphering the molecular mechanisms underlying their cross-regulation is essential to uncover the complex dynamics of host-pathogen interactions. Advanced investigations, including molecular and cellular studies, can shed light on the reciprocal regulation between HIFs and NF-kB, enabling the development of therapeutic strategies that target both pathways synergistically ( 68 , 69 ). In addition, advanced investigations should explore the functional consequences of modulating HIFs on host immune responses. HIFs can modulate immune cell function, cytokine production, and anti-microbial responses, thereby influencing the outcome of infections. Elucidating the impact of HIFs modulation on immune cell behavior, including phagocytosis, antigen presentation, and T cell responses, is critical for understanding the immunological consequences of targeting the HIFs pathway. This can be achieved through advanced immunological techniques, such as flow cytometry, functional assays, and in vivo models ( 70 , 71 ). Moreover, advanced investigations should incorporate systems biology approaches to gain a comprehensive understanding of the complex molecular networks involved in infectious diseases. Integrating multi-omics data, including genomics, transcriptomics, proteomics, and metabolomics, can provide a holistic view of the host-pathogen interactions including signaling cascades for different pathogens. These research along with computational analyses can unravel the intricate molecular pathways and identify key nodes or modules that can be targeted for therapeutic intervention ( 72 , 73 ).

Developing pharmaceutical HIF-1α agonists to inhibit the microbial activity of pathogens in different cell types could be an exciting strategy for supplementary therapy against complex infections caused by antibiotic-resistant pathogens or treating infections in patients with compromised host immunity. As aforementioned, HIF-1α is increasingly being investigated as a potential target in the fight against bacterial, viral and parasitic infections, particularly multidrug-resistant ones. Modulating HIF-1α as a pharmacologic intervention for treating chronic inflammatory disorders or improving innate immune function has been exhaustively proposed ( 58 ). Moreover, inhibiting HIF-1α activity may be a promising therapeutic approach for sepsis triggered by LPS ( 74 ). To develop effective therapeutic strategies for infectious and inflammatory diseases, a solid grasp and understanding of the underlying biology is required ( 75 ). First, there is a need to identify novel genes and targets specific to various pathogens which can be modulated directly or indirectly by HIF-1α inhibition. Table 1A provides a list of genes that have been tested for various pathogens in the context of HIF-NF-kB crosstalk and therapeutic development. Moreover, in general terms, comprehensive research efforts are required to uncover specific molecules, receptors, and signaling pathways that play crucial roles in the establishment of Infection and evasion of host immune responses. Identifying these novel targets would enable the design of more precise and targeted therapeutic interventions. Second, an important point for consideration in therapeutic design is usually having a grasp of the dynamics of the pathogen’s variability. Infectious agents exhibit genetic and phenotypic variability, which can influence their virulence, drug resistance, and ability to evade host immune responses ( 75 ). Therefore, investigating the molecular mechanisms underlying pathogen variability and its impact on disease progression and therapeutic outcomes could be germane in this context. This includes studying genetic diversity, mutations, and genetic exchanges among pathogens, as well as characterizing their phenotypic changes and adaptation to different host environments (either in normoxia and hypoxic cellular environment). Additionally, as mentioned above, the integration of multi-omics approaches is necessary to gain a comprehensive understanding of the molecular pathways involved in infectious diseases. Utilizing genomics, transcriptomics, proteomics, and metabolomics can provide insights into the complex interactions between host and pathogen ( 75 ). Data garnered from these advanced approaches, together with computational modeling and network analysis could provide a robust understanding of the effect of the HIF-NF-kB crosstalk on infectious disease processes. This integrative approach is vital for obtaining a holistic view of the underlying biology and identifying novel therapeutic targets. Lastly, it is crucial to validate findings from preclinical studies and translate them into clinical applications. While in vitro and animal studies provide valuable insights, it is essential to validate the efficacy and safety of potential therapeutic strategies in human subjects. Table 1B shows a list of current HIF-1α inhibitory or upregulating molecules and drugs currently under clinical trials. These studies would ideally involve conducting well-designed clinical trials to assess the therapeutic potential of identified targets. For this, physiologically relevant disease models can be employed to answer the needed research questions. In this regard, novel animal models such as the zebrafish, in combination with traditional cell lines and murine models, could help to elucidate the extent of the effects of HIF modulation on cellular physiology ( 157 ). We strongly believe targeting HIF-NF-kB crosstalk holds promise for therapeutic interventions for various microbial infections. Studies employing genetic and chemical inhibition of the PHD proteins have demonstrated therapeutic benefits in various disease models, such as colitis, where inflammation and NF-kB activity are significant contributors ( 158 ). Therefore, it would be intriguing to investigate the potential therapeutic impact of HIF activators and inhibitors on other infectious agents. More research efforts are needed to test the efficacy of available HIF modulators in different disease models, which could provide valuable insights into the various forms of HIF-NF-kB crosstalk that occur in different cell types in the context of individual pathogen. Collectively, a progressive understanding of the interplay between HIF-1 and NF-κB pathways through hydroxylation ( 159 ) can lead to the development of novel therapies for infectious and inflammatory disorders. Table showing the effects of hypoxia on genes of various infectious agents. Table showing a list of HIF-NF-kB modulators highlighting their mechanism of action, accompany Clinical trial numbers (NTC), and related pathogen/diseases.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Conceptualization: OCA. Figure and Table: OCA, SS, SCO, RA, ROA. Article draft and review: All authors contributed to the writing and review of the manuscript.