Deciphering the Neurosensory Olfactory Pathway and Associated Neo-Immunometabolic Vulnerabilities Implicated in COVID-Associated Mucormycosis (CAM) and COVID-19 in a Diabetes Backdrop—A Novel Perspective

Recent Mucorales-mediated outbreaks of infections and an association of fungal infection with COVID-19 cases, as observed for COVID-19-associated mucormycosis (CAM), have posed new challenges for the management of patients in critical care units. Diabetes and hyperglycemia are integrally linked to the severity of COVID-19, and uncontrolled diabetes mellitus and COVID-19 have recently been (independently or in combination) associated with the emergence of aggressive mucormycosis due to attendant defects in innate immune recognition pathways. Therefore, the identification of novel global cellular stressors upregulated during diabetes to understand the contribution of diabetes-associated metabolic vulnerabilities can help build a Metabolic-Stress-Associated Interactome (MSAI). This interactome can help reshape the metabolic inflammation (meta-inflammation) underlying the clinical manifestations of COVID-19 to facilitate the rational design of effective therapies for COVID-19 and CAM. Accordingly, an important area of research in COVID-19 therapeutics is engaged with identifying diabetes-associated pan-cellular stressors to understand their role in immune deregulation during COVID-19 and CAM, including investigating the distant trans-neuro-vascular–endocrine axis’s role in coordinating cellular-stress recognition, transmission, compensation, and decompensation during inter-organ regulation of metabolic homeostasis in diabetes. We reviewed clinico-pathological and laboratory data to propose potential diabetes-linked novel neo-vulnerabilities that can reshape the olfactory mucosal immune landscape during airway infections such as COVID-19 and CAM.


Current Taxonomy of Mucorales and Congeners
The Mucoromycota phylum comprises the Mucormycotina, Glomeromycotina, and Mortierellomycotina sub-phyla that fall into the Mucorales, Umbelopsidales, and Endogo-In the above-described subset of diabetic patients, a potentiated inflammatory state is caused by the continuous and persistent recruitment of local immune cells such as macrophages and neutrophils, which in turn recruit a variety of inflammatory cytokines [17]. This phenomenon, which paradoxically switches yet another robust inflammatory phenotype when SARS-CoV-2 is positively flagged in these subsets of patients, predisposes patients to a variety of secondary infections including mucormycosis, such as COVID-19associated pulmonary aspergillosis (CAPA), disseminated fusariosis, and invasive candidiasis. The first was extremely devastating during the second wave of COVID-19 in the Indian subcontinent and resulted in a dual-disease term called CAM. Much of the immunopathogenesis of Mucorales in DM patients comes from the prototype species Rhizopus spp. [29].
Secondary infections in these subsets of patients caused by Mucorales are mediated via the hyper-secretion of pro-inflammatory cytokines by immune reactive cells that open up divergent pathways leading to a vicious cycle of CAM and the widespread insult of the host tissues. Moreover, a plethora of interventions in the management of COVID-19 have further potentiated serum ferritin and free iron levels, drastically restricting the efficiency of macrophage immunity. Evidently, through siderophores, Mucorales efficiently acquire free iron from patients with DKA, as higher serum levels of free iron result in acidosis and poor binding to proteins. Secondly, in a setting of uremia wherein desferrioxamine chelation may act as a source (similarly to a COVID-19-induced hyperglycemia (indiscriminate use of CS) and chronic inflammatory state), Mucorales facilitate the pivotal mechanism in the immunopathogenesis of CAM [6,7,29].
Two host cell receptors-the ACE2 receptors (which are expressed in abundance in host endothelium) and glucose-regulated protein 78 (GRP78) (in addition to its role in MAD) as a co-receptor by recognizing the spike protein (S) of SARS-CoV-2 and efficiently internalizing the virus into the host cells [30]-are also involved in endothelial cell barrier disruption, inflammation, coagulation, endotheliatis, and apoptosis. In severe cases of COVID-19, abnormally elevated levels of D-dimer, fibrinogen, and Willebrand factor (VWF) and profound increases in inflammatory cytokines such as IL-1, Il-6, IL-8, and TNF-alpha are indications of multi-organ dysfunction due to widespread venous thrombosis, systemic vasculitis, and vascular coagulopathy [29]. Rayner et al. found that the suppression of GRP78 expression resulted in the inhibition of SARS-CoV-2 replication [31]. The preexisting hyper immune-reactiveness, endothelial damage, and multi-organ dysfunction in ongoing COVID-19 are important risks for the emergence of complications owing to angioinvasive Mucorales [7,29,30] Recently, Franco et al. reviewed host-pathogen and molecular interactions in the pathogenesis of Rhizopus spp. in DM patients. Mucormycosis has six main clinical presentations, of which ROCM is the most predominant [3]. Though the occurrence of ROCM was preponderantly seen in immunocompromised patients in the pre-COVID-19 era, CAM has since created a similar niche by mimicking immunocompromised (IC) status in the present pandemic situation [32]. Augmenting this scenario, there have been multiple reports of mucormycosis in immunocompetent hosts, presenting as chronic ROCM, skull base invasion, and renal mucormycosis caused by these seemingly thought opportunistic invaders [33,34]. Regardless of presentation, the angioinvasiveness, dissemination to the contiguous tissues resulting in necrotic eschars and thrombosis along with mucoralean spores in IC hosts with notably high mortality rates [3]. Coincidentally, the angioinvasive process and thrombotic microangiopathy in the disease spectrum are the mimickers of both COVID-19 and mucormycosis pathogenesis. Furthermore, the striking mortality of mucormycosis in DM patients is as high as 77% due to infections by Cunninghamella bertholletiae, followed by 57% due to Rhizopus spp. and 41% due to Mucor spp., which indicates the need for the efficient management of these rapidly progressive conditions [35].
The spread pathways of Mucorales in ROCM were described by Hosseini and Bhorgei in 2005 [36]. In the inhalation mode, the pterygopalatine fossa serves as the main reservoir for mucormycosis, and the spores and hyphae lodge in the nasal passages and contiguous sinuses. GRPs are a group of immunoglobulin-binding proteins (BiPs) that include GRP78 (a molecular chaperone belonging to the heat shock protein 70 (HSP70) family located in the lumen of the endoplasmic reticulum) and its isoform GRP78va (located in the cytosol), which have pivotal roles in folding, assembly, and quality control of proteins and misfolded protein degradation. Furthermore, they are expressed in diverse types of inflammatory, cancerous, viral, and invasive fungal spores including the Mucorales [37,38]. Hyperglycemia-induced stress in Rhizopus spp. infection in DM patients results in the translocation of GRP78 onto the cell surface (cs) as csGRP78 and the overexpression of receptors for signaling, also leading to anti-csGRP78 autoantibodies. With the expression of fibronectin and type I collagen, fibrosis is a consequential event of the sequential interactions of csGRP78 with β1 integrin and the activation of the focal adhesion kinase (FAK) and downstream protein kinase Akt (van). Interestingly, a similar mechanism of receptor signaling and entry of the pathogen into the host cell was proposed for Dengue, Ebola, Coxsackie, and SARS-CoV2 viruses, along with Rhizopus spp. [39,40].
Receptor interaction studies of nasal and alveolar epithelia with homologous spore coating proteins (coat protein homolog (CotH) present on Mucorales such as Rhizopus delemar and Rhizopus spp. have revealed that csGRP78 and integrin-β1 are only overexpressed in the nasal epithelia of DM patients. This complex microenvironment, holding high glucose, iron, and DKA levels, promotes superficial tissue level invasion by favoring the interactions between CotH (specifically, CotH3) on Mucorales with that of the abundant csGRP78 and integrin-β1 on the nasal epithelium. Furthermore, this expands the virulence of Rhizopus spp. via coating with laminin and collagen IV potentiating the invasion, endothelial damage, and continuous expression of GRP78 in the contiguous tissues planes and endothelium, eventually establishing the clinical entity ROCM in DM [36][37][38][39].
Beguilingly, Mucorales have adapted to the novel acquisition of virulence factors by harboring endosymbiotic nosocomial Gram-negative bacteria including various species of Burkholderia and Ralstonia (R.) pickettii. The former releases potent toxic metabolites called rhizoxins by endosymbiosis, and the latter plays a role in the intracellular survival of macrophages and triggers a profuse pro-inflammatory cytokine release [41,42]. Intriguingly, R. pickettii endosymbiosis with Rhizopus microspores has complicated several nosocomial outbreaks of Mucorales due to their propensity to contaminate sterile water, saline medical solutions, and hospital disinfectants [40,42].

The Complex Interplay of Various Factors: Mucosal Proteases and Iron Redox Stress
Human environments, whether community settings, indoor or outdoor areas, or airconditioned or non-air-conditioned hospital areas, allow mucoralean spores to become airborne and inhaled by immunocompetent hosts without any apparent clinical disease. However, in an event of a source with a high hyphal burden, the polymorphoneutrophils (PMNs) response is overwhelmed, thus surpassing the primary barriers of immunity. The usual modes of transmission are inhalation, ingestion, or direct inoculation. The size of sporangiospores may also contribute to their role in establishing human disease, as larger R. arrhizus spores tend to settle in the upper respiratory tract and the smaller Cunninghamella or R. microsporus spores reach the lower respiratory tract; the latter is highly virulent and leads to rapid disease [6,43,44]. Furthermore, molecular and epidemiological studies to delineating the mechanisms of acquisition of hypervirulence, Mucorales genus-specific differences in interactions with the host cells (GRP78 expression and CotH3 interactions), the role of SARS-CoV-2 in potentiating diseases leading to CAM, other co-infections, and nosocomial outbreaks in increased CAM cases in aggravating seemingly opportunistic Mucorales infection are currently underway [6,43,45].

Expanding the MSAI-Proteolytic Stress as a New Player in COVID-19 Arena
The potential diabetes-associated cellular-stress pathways emerging from multi-faceted high throughput omics approach are facilitating expansions on MSAI and the development of the understanding of the pathogenesis of beta-cell dysfunction in COVID-19, which is incompletely understood even though integrated stress response (ISR) is integral to the development of diabetes. The impairment of insulin-producing pancreatic beta-cells is a key contributing factor to the diabetic pathogenesis by SARS-CoV-2. However, whether beta-cells are damaged by the coronavirus through a direct action is less clearly understood. The pancreatic cells are governed by intricate inter-organ network regulation involving soluble humoral factors from diverse organs that crosstalk with their neuro-vascular conduits. Therefore, linking the cause of pancreatic beta-cell damage in subjects with COVID-19 to the direct infection of SARS-CoV-2 or alternate indirect effects including distant organ damage, heralding inflammatory loops, and yet-unexplored cellular stressor(s) remains challenging. However, the potential of direct infection with SARS-CoV-2 and the subsequent cell fate reprograming of pancreatic cells has been recently demonstrated [46][47][48]. Notably, heterogenous host pancreatic (endocrine, exocrine, and acinar) cellular-stress responses caused by SARS-CoV-2 infections have displayed the transdifferentiation of insulin-producing beta cells into glucagon-producing alpha and trypsin-producing acinar cells in the pancreas, which has been linked to an ISR pathway. Remarkably, a trans-ISR inhibitor treatment was found to increase insulin expression and reduce glucagon, trypsin, and cell stress-associated genes in SARS-CoV-2-infected human islets [47], indicating a possible role of co-secreted pancreatic trypsin/proteolytic stress in reshaping diabetes-associated ISR.

Enhanced SARS-CoV-2 Transmissibility and Role of Olfactory Mucosal Proteases
Viral proteases are reported to have potential ancillary functions extending beyond the polyprotein/spike clipping that are linked to host immune evasion [49]. However, the biological functions (outside fusogenic potential) of cellular host proteases activated during COVID-19 remain largely unexplored. Importantly, the TMPRSS2 and trypsin serine proteases have been identified as important molecular players in the underlying pathology and emergence of coronaviruses, respectively. In particular, the trypsin treatment (proteolytic processing) of viral spike proteins has been identified as a potential species barrier for the emergence of zoonotic coronaviruses, hence posing a potential threat for future outbreaks driven by the cross-species transmission of coronaviruses [50][51][52][53][54]. It was proposed that additional small intestine proteases such as trypsin could enhance SARS-CoV-2 s viral infection and pathogenesis capability by triggering more robust cell fusion [55]. The increased transmissibility of SARS-CoV-2 compared to SARS-CoV is linked to the occurrence of a unique furin-cleavable polybasic motif (RRAR) at the S1/S2 boundary, as the cleavage results in a C-terminally exposed RRAR peptide that is capable of binding to neuropilin-1 (NRP1), which has been demonstrated to be an important host factor receptor for SARS-CoV-2 by facilitating entry and infectivity [50,56,57]. The novel core region with the SPRRAR ( S 680-V 687) polybasic insert of SARS-CoV-2 harbors positively charged arginine at 683 and hydrophobic alanine at 684, which makes the site susceptible to promiscuous binding/cleavage by not only serine proteases such as furin or furin-like PCs but also mono-and dibasic amino acid targeting serine proteases such as matriptase, human airway trypsin, TMPRSS2, and kallikrein. Importantly, a plethora of serine proteases (SPs) such as furin, TMPRSS2, furin-like PCs, and trypsin-like proteases in the nasal microenvironment have facilitated the heightened transmissibility of emerging SARS-CoV-2 variants that have enhanced susceptibility to SP-cleavable polybasic amino acids; for instance, a mutation of non-polar proline at 681 to more positively charged arginine in delta variant (P681R) in spike protein resulted in enhanced transmissibility due to more cleavability at S1/S2 [58,59], and a mutation of proline to histidine (P681H) in omicron variant's spike protein [60] was recently reported and affirmed the trend of enhanced proteolytic susceptibility and concomitant high transmissibility. The nasal serine proteases are therefore integrally linked to the increased infectivity of SARS-CoV-2 due to indispensable prerequisite of proteolytic-priming of spike protein by diverse nasal SPs. It is a reasonable speculation that emerging variants of concern will display increased infectivity and transmissibility due to the subsequent shedding of biologically functional cleaved fragments that tend to retain the potential to not only bind olfactory cells (e.g., RRAR binding to NPR-1) but are also secreted into the mucous and exported as respiratory droplets to transmit infection, as has been previously reported [57].

Neuro-Vascular Olfactory Mucosal Niche in Diabetes and SARS-CoV-2 Pathogenesis
The neurological symptoms of COVID-19 and long COVID including anosmia, headache, encephalitis, and neurovasculopathy are becoming increasingly recognized (reported in up to 85% of ICU patients); however, neuroinvasion/neurotropism or the presence of SARS-CoV-2 in olfactory neurons and the brain remains unproven [61][62][63][64]. The transport of viruses through the neuro-vascular niche of olfactory mucosa all the way to CNS is debatable, but damage to neuro-vascular immune units of the brain comprising neural-crestderived vascular pericytes and neuroglial astrocytes that participate in neuro-inflammation is more accepted [61,65]. The most well-documented step in the pathophysiology of SARS-CoV-2 is the primary route of infection, the olfactory mucosa, wherein the nasal-ciliated cells and non-neuronal sustentacular cells are the primary targets of the virus replication during the early stages of COVID-19 [62,66].
Interestingly, though anosmia and olfactory dysfunction are hallmark and consistent neurological symptoms of COVID-19, mechanistic insights of how neurosensory perception is altered and the involvement of the olfactory-nerve-mediated transport of viruses to the CNS are unresolved [67][68][69][70]. Despite the abundant expression of viral entry proteases (ACE2 and TMPRSS2) in human and mouse olfactory systems and indications of sustentacular cells as prime targets of SARS-CoV-2 [71][72][73], viral replication in the non-neuronal olfactory epithelia of patient samples could be demonstrated only recently [62] due to the technical challenges of accessing the olfactory epithelium (OE) during active infection.
It is noteworthy that the major target sustentacular cells (SNCs) are glia-like in functionality. Given the paucity of literature on human SNCs, comparisons to animal models are challenging; nevertheless, in a rat model, SNCs were proposed to be glucosetransporting cells from the blood across the apical mucus to fuel olfactory sensory neuron (OSN) cilia [74,75]. Additionally, SNCs have been implicated in sheathing OSNs [76,77], which suggests a highly supportive role of SNCs in OSNs survival and maintenance, and any dysregulation in these supporting cells could lead to olfactory transduction pathway defects such as anosmia. Furthermore, the existence of a closely apposed olfactory neurovascular unit (not much explored) could also underlie neuro-inflammatory events resulting in massive immune dysregulation in the upper respiratory pathway during COVID-19 and paving the path for the aggressive presentation of mucormycosis. Investigating the odorant receptor repertoire is yet another potential area to inform on any association with inflammatory networks, but it is challenging to map receptor expression in COVID-19active subjects during the acute phase of the disease marked by anosmia; nevertheless, the olfactory epithelia of deceased patients did not show any differences [62]. The strong correlation of diabetes to COVID-19 and CAM/ROCM infection suggests potential "proviral/pro-fungal" cellular host factors in OE, which could be linked to glucose homeostasis deregulation and immune derangement in the olfactory mucosae of diabetic patients, hence explaining the predisposition of upper airway infections in diabetic subjects. This premise may be supported by a recent study that reported the identification of a UGT2A1/UGT2A2 single locus gene encoding for UDP glucuronosyltransferase enzyme in COVID-19 patients with anosmia [78], thereby reemphasizing the involvement of a glucose stress-associated anomalous glycosylation process that is integrally linked to disturbances in mucosal homeostasis and inflammation [79]. The possible role of glycosylation in altering the olfactory mucosal immune landscape is discussed in a later section. We first discuss the association and role of proteases in inflammatory pathways triggered by protease-activated signaling cascades in infectious inflammatory diseases.

Proteases as Signal Transducers-A Role beyond Spike Clipping
Olfactory infection and dysfunction occur early on during COVID-19, overlapping with severe invasive fungal co-infections including ROCM in primarily hyperglycemic patients with compromised immune status, and CAM was recently characterized by an unprecedented upsurge in fungal cases during a delta variant outbreak in India. Given the high proteolytic priming requirement and increased infectivity of the delta variant (as discussed above), it is reasonable to propose that a protease-rich neuro-immune unit of olfactory mucosa could play a crucial role in the rapid spread and systemic dissemination of fungal infections via neuro-inflammatory responses mediated by dysregulated olfactory transduction pathways, thereby compromising the anti-fungal immunity in diabetic patients with COVID-19 during ROCM infection. Nevertheless, any strong correlation between host protease-mediated immune dysregulation has not been reported for COVID-19 so far. However, the majority of the serine proteases in OE have been reported to have cognate signaling receptors, and many such protease-receptor signaling pathways have been established for other microbial diseases.
Emerging paradigms suggest an increased trend towards "trypsin-reliance" for zoonotic coronavirus emergence that might predict the cross-species transmission of coronaviruses possibly employing alternative route(s) such as the digestive tract/gut, which has been proposed to be a potential site for future coronavirus emergence events in humans, and alternative but unidentified ACE2-independent host-cell receptors [53,54,80,81] that continue to be reported with broader host-receptor repertoire linked to an enhanced infectivity and transmissibility of SARS-CoV-2 [58]. The ACE2-independent or partially dependent entry of SARS-CoV-2 is becoming increasingly recognized by studies supporting the hypothesis that host cells infected by SARS-CoV-2 morph into virus-permissive cells that preset low ACE2 expression [82], indicating lower ACE2 thresholds for successful infection in some tissues or the presence of alternative receptors for viral entry. SARS-CoV-2-infected trachea transcriptomic signatures were found to parallel hematopoietic lineage progenitor cells, indicating a possibility of hematopoiesis induction by SARS-CoV-2 [83]. These paradigms are suggestive of potential mechanisms that might be active at the trypsin (protease)-host interface, which may extend beyond the layer of "viral-spike processing" and could be linked to "host-receptor alterations", as previously proposed [54].
Recent findings from diverse groups support the "co-emergence/-existence of SARS-CoV-2 infection and cell fate reprogramming events", indicating extended SARS-CoV-2associated reprogramming trajectories including possible hematopoiesis, [83] preferred enterocyte progenitor (low ACE2) permissive replication [82], and transit-amplifying cells or intestinal stem cells supporting SARS-CoV-2 infections [84]. Furthermore, recent studies on SARS-CoV-2 infected olfactory mucosa reported that the infected nasal epithelial cells were subsequently regenerated by basal stem or other niche cells [62,66], which has been also reported for the regeneration of trachea, intrapulmonary airways, and alveoli in COVID-19 subjects [85]. The incorrectly timed growth factor responsiveness has been linked to disease severity [86], and tissue reparative growth factor signatures have been linked to recovery from moderate COVID-19 [87], thus indicating orchestrated spatiotemporal wound healing to be an important component in determining the extent and severity of COVID-19.
The regulation of the protease/antiprotease balance during pathologic insults (marked by the up-or downregulation of serine protease/SERPIN) is very complex and underlies tissue integrity and inflammation via various growth-factor-linked signaling pathways. Type II transmembrane serine proteases such as matriptase and kallikreins are known to activate protease-activated receptor-2 (PAR-2) and be regulated by SERPIN SPINT1/HAI-1 [88]. Recent studies have implicated coagulation proteases such as TF, FVIIa, and FXa in the activation of signaling receptor PAR-2, which is linked to pro-viral responses via TLR3 [89][90][91][92][93][94][95][96][97]. The authors of recent study proposed that targeting FXa and thrombin by using specific anticoagulant drugs can inhibit PARs, which may have beneficial role in human inflammatory diseases [97]. PAR-2 signaling can also be triggered by trypsin, tryptase, neutrophil elastase, GPI-anchored serine protease (PRSS8) prostatin, membraneanchored hepsin, and TMPRSS2. PAR-2 targeting can have beneficial effects in the context of infectious pathology; notably, PAR-2-mediated NETosis was recently linked to pathogen benefits [98].

In Vitro Disease Modelling to Recapitulate COVID-19 and Diabetic Pathways
Given the potential of invoking inflammatory signaling cascades by serine proteases, as encouraged by our previous findings [99][100][101][102], we speculated that proteaseantiprotease disbalance could induce concomitant metabolic reprogramming events driving viral-competent neuro-immunomodulatory pathways. We recently tested a potential hypothesis regarding whether proteolytic stress (proteolytic activity associated with COVID-19-activated TMPRSS2, furin, complement coagulation serine proteases, DPP4, HAT, neutrophil elastase, etc.) can upregulate metabolic stress and intensify metabolic reprogramming, thus resulting in altered neuro-immune responses in monocytic cells. Neural-crest-originating astrocytes and pericytes are becoming increasingly appreciated responder cells underlying neurovascular abnormalities in COVID-19 pathobiology. We explored whether developmental epithelial-mesenchymal transition (EMT) events initiated by active proteases during neural-crest generation could be recapitulated by providing exogenous proteolytic exposure to pathology competent cells in vitro, resulting in the upregulation of developmental multipotent neural-crest-like cells with ectomesodermal potential capable of upregulating neurogenesis/gliogenesis (neurons and astrocyte) and vasculogenesis (microvascular pericyte), crucial cellular processes underlying neuroinflammatory diseases [Sharma et al., unpublished data].

In Vitro Virus-Free Model-Establishment and Characterization of a Novel Proteolytically Tunable Plasma Based Cellular Stress Model for COVID-19 Modeling
A novel in vitro virus-free model of COVID-19 was designed to investigate the effect of serine protease trypsin as a potential cellular stressor on a disease-competent monocytic THP-1 cell line that could result in COVID-19-associated pathways upon transcriptomic profiling. The cellular model was a slight modification of our previous findings [102], as we incorporated healthy plasma in the current model in addition to the use of serum-free media and the testing of escalating doses of trypsin. Briefly, monocytic THP-1 cells were cultured on a combination of serum-free media and plasma (control, C); THP-1 cells cultured on control/C supplemented with low trypsin (30 µM) were named low proteolytic stress (LPS), and THP-1 cells cultured under control/C supplemented with high trypsin (100 µM) were named high proteolytic stress (HPS). Finally, we evaluated the growth factor responsiveness of THP-1 cells in the presence of 100 µM of trypsin. Briefly, adult retinal pigment epithelial (ARPE-19) cells were cultured in 100 µM of trypsin in serum-free media at a density of 1 million cells/mL for 72 h. The condition media were collected and used to treat THP-1 cells for a further 72 h. The ARPE-19-conditioned media well-characterized by us and is marked by the secretion of biologically active bFGF (basic fibroblast growth factor) and interleukin IL-1ß, as determined with multiplex assays [99][100][101]. The ARPE-19-conditioned media therefore contained bFGF, IL-1ß and active trypsin (as qualitatively determined by gelatin zymography-data not shown), but absolute concentrations of trypsin were not determined with a commercial trypsin substrate. We named this group HPS-GFC (high proteolytic stress growth factor cytokine). The THP-1 cells cultured under trypsin conditions were incubated for 72 h at 37 • C and 5% CO 2 . Each experiment was conducted in triplicate.

Transcriptomic Profiling
RNA sequencing was outsourced to Redcliffe Life Tech, Noida, India, which performed transcriptomics using an NGS Illumina platform with a 150 read length and paired end sequence layout. Quality control was carried out using the fastp tool (0.20.1) to provide clean data for downstream analysis. NGS sequencing reads were mapped with the Hisat2 (2.1.0) alignment tool. For abundance estimation, the data input was subjected to the Counts (2.0.1) tool in either the Sequence Alignment Map or Binary Alignment Map format. Differential gene expression and visualization were carried out using the DeSeq2 (1.8.3) tool. Proteolytic stress was tested to assess the upregulation of COVID-19-relevant inflammatory pathways using unbiased transcriptomic profiling.
Gene Set Enrichment Analysis ridge plots were used for significant KEGG terms. Ridge plots are density plots of the frequency of log2 fold-change values per gene within each enriched KEGG group, which helps to interpret the up-or downregulation of that KEGG category. Here, plots were created in clusterProfiler using KEGG orthologue annotations and log2 fold changes per gene calculated by DESeq2 during differential expression analysis. X-axis is the log2 fold change in expression for genes present in each plotted KEGG category, with positive values indicating upregulated expression in replicates and negative values indicating downregulated expression in replicates. Peaks are colored by corrected p-value, as shown by the legend, and corrected pand q-values are shown per KEGG category.
are shown per KEGG category.

Results
Interestingly, the monocytic cells upregulated COVID-19-associated pathological pathways upon escalating proteolytic stress while concomitantly upregulating metabolic stress, suggesting the importance of ensuing proteolytic stress underlying COVID-19 pathology. The combined role of metabolic and proteolytic stress resulted in the upregulation of COVID-19 pathways that co-segregated with complement-coagulation, olfactory transduction, steroid biosynthesis, iron-ion binding, ferroptosis, and maturityonset diabetes (Figures 1-5).  The interferon response pathway genes were also found to be overexpressed under LPS conditions (data not shown), suggestive of a mild-COVID-19-like disease phenotype. The HPS condition was characterized by upregulation of COVID-19 pathway along with complement-coagulation, metabolic, neurodegenerative, and several bacterial and viral infectious pathways, thus indicating a hyper inflammatory or severe COVID-19-like disease pathway in vitro. HPS was found to result in concomitant upregulation of metabolic stress, which may have implications in pathophysiology of COVID-19 as a plethora of trypsin-like serine proteases including TMPRSS2, furin, DPP4, complement-coagulation serine proteases, neutrophil elastase, and trypsin are upregulated during the inception and course of the disease. . The LPS pathway network indicates upregulation of antigen processing and presentation, thyroid hormone synthesis, and COVID-19 pathway upregulation. The interferon response pathway genes were also found to be overexpressed under LPS conditions (data not shown), suggestive of a mild-COVID-19-like disease phenotype. The HPS condition was characterized by upregulation of COVID-19 pathway along with complement-coagulation, metabolic, neurodegenerative, and several bacterial and viral infectious pathways, thus indicating a hyper inflammatory or severe COVID-19-like disease pathway in vitro. HPS was found to result in concomitant upregulation of metabolic stress, which may have implications in pathophysiology of COVID-19 as a plethora of trypsin-like serine proteases including TMPRSS2, furin, DPP4, complement-coagulation serine proteases, neutrophil elastase, and trypsin are upregulated during the inception and course of the disease.         in our in vitro disease modelling. A regulated IL-1 beta/inflammasome activation pathway can have beneficial effects in controlling COVID-19. Transcriptomic profiling data of monocytic THP-1 cells treated with prototypic serine protease trypsin (proteolytic stress) at a low and escalating concentrations are represented as low proteolytic stress (LPS) and high proteolytic stress (HPS) compared to control/C, respectively. HPSGFC condition is depicted in the extreme right under the heading "proteolytic stress + growth factor responsiveness". The addition of neuroregenerative growth factor to the high proteolytic environment is speculated to ameliorate COVID-19, but the upregulation of cortisol secretion can result in development of T2DM and post-COVID mucormycosis, even in the presence of the sufficient secretion of insulin. Olfactory transduction can be linked to both COVID-19 development and downregulation depending on the presence of growth factor responsiveness. We present the differentially regulated significant biological pathways under various treatment groups, data collected with the extended GO biological pathway enrichment analysis tool (data not shown). We use the term "neuroinflammation" to link co-expression of olfactory transduction and coagulationcomplement pathways, and we hypothesize that neurosensory olfactory transduction can keep immune responses under check and limit hyperinflammation in presence of soluble growth factors and cytokines, as in HPSGFC conditions. Endocrine and neuroendocrine reshaping is also associated with growth factor and cytokine responsiveness. Therefore, olfactory dysfunction or anosmia alone may not be strong predictors of the course of COVID-19. Additional parameters such as growth factor assessment, proteolytic activity and neuroendocrine peptides in the plasma and nasopharyngeal swabs could help improve stratifications of disease severity.
Furthermore, comparisons of diverse treatments allowed us to classify groups that displayed the following: (a) the COVID-19 pathway along with the co-expression of upregulated interferon response, oligoadenylate synthetase, double-stranded RNA binding, and innate immune recognition pathways and a downregulated olfactory transduction pathway, thus suggesting a mild-COVID-19-like pathological response; (b) COVID-19 pathway co-segregating with upregulated complement coagulation, acute inflammatory response, and steroid biosynthesis pathways, thus suggesting a severe COVID-like pathological response; and a unique group (c) a downregulated COVID-19 pathway co-segregated with upregulated olfactory transduction, an RNA interference Transcriptomic profiling data of monocytic THP-1 cells treated with prototypic serine protease trypsin (proteolytic stress) at a low and escalating concentrations are represented as low proteolytic stress (LPS) and high proteolytic stress (HPS) compared to control/C, respectively. HPSGFC condition is depicted in the extreme right under the heading "proteolytic stress + growth factor responsiveness". The addition of neuroregenerative growth factor to the high proteolytic environment is speculated to ameliorate COVID-19, but the upregulation of cortisol secretion can result in development of T2DM and post-COVID mucormycosis, even in the presence of the sufficient secretion of insulin. Olfactory transduction can be linked to both COVID-19 development and downregulation depending on the presence of growth factor responsiveness. We present the differentially regulated significant biological pathways under various treatment groups, data collected with the extended GO biological pathway enrichment analysis tool (data not shown). We use the term "neuroinflammation" to link co-expression of olfactory transduction and coagulation-complement pathways, and we hypothesize that neurosensory olfactory transduction can keep immune responses under check and limit hyperinflammation in presence of soluble growth factors and cytokines, as in HPSGFC conditions. Endocrine and neuroendocrine reshaping is also associated with growth factor and cytokine responsiveness. Therefore, olfactory dysfunction or anosmia alone may not be strong predictors of the course of COVID-19. Additional parameters such as growth factor assessment, proteolytic activity and neuroendocrine peptides in the plasma and nasopharyngeal swabs could help improve stratifications of disease severity.
Furthermore, comparisons of diverse treatments allowed us to classify groups that displayed the following: (a) the COVID-19 pathway along with the co-expression of upregulated interferon response, oligoadenylate synthetase, double-stranded RNA binding, and innate immune recognition pathways and a downregulated olfactory transduction pathway, thus suggesting a mild-COVID-19-like pathological response; (b) COVID-19 pathway co-segregating with upregulated complement coagulation, acute inflammatory response, and steroid biosynthesis pathways, thus suggesting a severe COVID-like pathological response; and a unique group (c) a downregulated COVID-19 pathway co-segregated with upregulated olfactory transduction, an RNA interference pathway, and a TCR receptor pathway. We hypothesize a potential (novel) adaptive immune reshaping linked to olfactory transduction through a neuropeptide-signaling pathway that could reshape inflammatory responses by inducing antigen-specific T-cell responses by involving the RNA interference/RISC pathways of gene silencing and regulation. We are in the process of establishing the significance of these pathways in diabetic patients with COVID-19 and CAM [Sharma et al., unpublished work]. We earlier proposed pathogen-invoked proteolytic activation as a novel cellular stress-inducing process that could facilitate viral-competent immunomodulation through reprograming RNA metabolism and homeostasis in a process involving the protease-induced transcriptomic/epi-transcriptomic reshaping (PITTR) of host cells to counter cellular stress [102].
The pre-symptomatic or acute phase of COVID-19 marked by high viral loads/ replication in the nasal epithelium makes it a potential target to inhibit the intensification of infection by limiting spread of the infection. Therefore, the prophylactic/therapeutic targeting of the olfactory epithelium by employing intranasal sprays is envisioned as a potential approach to prevent COVID-19 severity. The use of intranasal drug formulations are highly encouraged in COVID-19 and CAM treatment due to initially higher viral loads in the nasal microenvironment and their direct detrimental impact on infection inception and clinical course. Recently developed imidazole compounds have been reported to enhance anti-viral interferon responses upon intranasal administration in animal settings [103]. Similarly, recent phase-II clinical trials from Canada and the UK, showed the efficacy of nitric oxide nasal spray (NONS) in reducing SARS-CoV-2 viral loads for treatment of COVID-19 infections [104,105]. We envision strong anti-diabetic and antimicrobial effects of the combination of anti-metabolic and anti-proteolytic (MPI) inhibitors. Metabolic stress/hyperglycemia intensifies systemic SARS-CoV-2 and nasal/olfactory ROCM spread. We expect a prophylactic use of the MPI intranasal formulations in diabetes patients that could exhibit beneficial effects in regulating ferroptosis, promoting iron homeostasis, and facilitating the management of COVID-19 and CAM due to its anti-diabetic, anti-inflammatory, anti-oxidant, and anti-chelating properties.

Hexosamine Biosynthetic Pathway of Glycosylation and Metabolic Stress Calibration in Olfactory Mucosa
Metabolic rewiring is a common mode of adapting to cellular stress to restore homeostasis due to its plasticity in cellular metabolism. As evidenced in uncontrolled hyperglycemia, persistent metabolic alterations can result in the irreversible resetting of the metabolic machinery, thus resulting in the desynchronization of the functional outputs. The primary bioenergetically tuned metabolic pathways include AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), the hexosamine biosynthetic pathway (HBP), and iron handling. Major cellular calibrants of energy stress include the innate immune myeloid lineage monocyte/macrophages and neutrophils that respond to metabolic stress with remarkable resilience by adjusting to distinct metabolic states by displaying phenotypic, transcriptomic, functional, and spatial heterogeneity, thereby diversifying the inflammatory immune responses. Accordingly, burgeoning metabolic diabetes disorders are linked to myeloid cell dysfunction, which has been recognized as important feature in SARS-CoV-2 infection. Diabetes and hyperglycemia have been integrally linked to the severity of COVID-19, and uncontrolled diabetes mellitus and COVID-19 have recently been independently or in combination associated with the emergence of aggressive mucormycosis [16,19,[106][107][108][109][110][111][112][113][114][115][116]. The induction of diabetes in SARS-CoV-2 infectious settings has also been demonstrated [47,48,117], suggesting an important role of metabolic resetting during COVID-19 disease course.
Importantly, infection-triggered (SARS-CoV-2/Mucorales) inflammasome-mediated pyroptotic cell death has been intricately linked to release of DAMPs and extracellular ATP, monocyte/macrophage activation, epithelial and endothelial cell damage, and vascular leakage, all resulting in inflammatory immune cell infiltration into the nasal/respiratory mucosa. The proinflammatory cytokines consequently lead to mucosal mucin reshaping and the activation of thromboinflammation. What remains underappreciated is the fact that the pathogenic microbes (SARS-CoV-2/Mucorales) have to traverse the bulky polyanionicmucus barrier (20 µm) and heparan sulfate proteoglycans (HSPGs) (50 nm), embedded in the cell membranes to reach and destroy the nasal/airway epithelial cells. The mucins (terminally charged sialic acid and hydrophobic fucose) and polyanionic HSPGs offer potential structural frameworks for mediating interactions with primary binding motifs in the infecting microbes (e.g., cationic RBD groove of SARS-CoV-2 and fungal lectins attach to mucins and HSPGs to facilitate binding and entry to the host cells). Following binding, the microbes can be inactivated/cleared-off by mucociliary clearance or be subjected to infection spread, depending on the cellular/extra cellular metabolic milieu. The antipathogenic and rheologically flexible properties of mucus come at the cost of the high energy consumed in the process of mucous hydration in the airway system, which is highly dependent on the pathologically overexpressed metabolites (alarmin/DAMPs) including extracellular ATP and UDP-sugars derived from the HBP pathway (glucose/galactose) [131][132][133][134][135]. Therefore, it is reasonable to hypothesize that diabetes-associated metabolic pathways participate in reshaping mucin biology and innate immunity in the upper airways, thus making them susceptible to infection and the spread of microbes.
It could be speculated, that T2DM-responsive bioenergetically tuned HBP deregulation alters the "metabolic glycan-code input"; as opposed to the primary metabolic sensing by myeloid "cellular sensors" such as macrophages/monocytes. This metabolic perturbation (altered glycosylation) underlying diabetes is first read by the innate immune "structural sensors", such as airway epithelial mucins and endothelial glycocalyx that are heavily glycosylated macromolecules. Epithelial mucins and endothelial glycocalyx are arranged as multiscale hierarchal structured fluids in nasal and respiratory epithelia, therefore acting as the frontline structural barricades of the mucosal innate immune system. The altered glycosylation patterns in nasal/respiratory mucosa and endothelial glycocalyx compromise epithelial-endothelial barrier integrity and offer easy pathogenic access to the underlying epithelium/endothelium, thus resulting in enhanced spread, enhanced vascular permeability, and thromboinflammation. Concomitantly, there is heightened purinergic signal transduction via the HBP intermediate UDP-sugar (ligand)-mediated activation of metabotropic purinoreceptor P2Y14 (expressed on adipocytes, myeloid immune cells, hematopoietic stem cells, kidneys, lungs, and intestines). The P2Y14 receptor was recently implicated as a strong target for neutrophilia attenuation in severe COVID-19 [132]. However, no data on COVID-19 or mucormycosis patients are available to date. P2Y14 expression is linked to glucose and lipid homeostasis [136], as well as the amplification of allergen-induced hyperinflammatory airway eosinophilia [135]. The HBP pathway has also been recently linked to ferroptosis sensitivity [137], thereby indicating a potential druggable "diabetes-HBP-mucin-iron axis" in COVID-19 and mucormycosis infections. Diabetes-associated, HBP-mediated altered glycosylation patterns in epithelial mucins and the role of the HBP-derived, UDP-sugar-activated P2Y14 purinoreceptor in myeloid immune deregulation remain subjects that have yet to be explored.

Diabetes and Metabolic Iron Redox-Stress-Macrophages as Ferrostats
It is important to note that the immune cells that act as primary metabolic sensors with high plasticity in metabolic switching (glycolytic vs. oxidative phosphorylation) are myeloid lineage macrophages, which are also central to anti-fungal/anti-Mucorales immunity. The longitudinal profiling of respiratory and systemic immune revealed myeloid cell-driven inflammatory events in COVID-19 [114]. The differential activation potential of NLRP3 inflammasome among myeloid cells is suggested to be a biomarker for the course of COVID-19 [115]. SARS-CoV-2 was shown to engage inflammasome and induced pyroptosis in monocytic cells [116]. Therefore, the metabolic reprograming of macrophages during hyperglycemia potentially alters the gene expression and regulation of immune pathways important for innate immunity and subsequent macrophage-sculpted adaptive immunity. Histone acetylation was recently reported to be upregulated in macrophages following concerted increases in glycolytic flux and ATP-citrate lyase activity [109]. Monocytopenia and morphological monocytic defects were found to be associated with hyperinflammation in COVID-19 in T2D patients [110], and hyperglycemia was shown to drive proinflammatory M1 polarization in macrophages via the TLR4-IRF5 pathway [111]. The defective macrophages thus fail to efficiently reshape the adaptive immune responses during inflammatory diseases such as COVID-19.
The enhancement of CD8 + T cell effector functions (shaping extra-follicular adaptive immune responses) and the identification of repertoire of neutralizing antibodies to offer protective immunity post-infection are intensive areas under research to prevent infection, re-infection, or superinfections. Persistent alterations of iron homeostasis/hyperferritinemia in COVID-19 are also associated with immune deregulation . Importantly, glycans/glycosylation is becoming increasingly relevant in shaping T cell effector functions and immunological synapses [163].
Macrophages are well-appreciated for their role in systemic iron homeostasis, in which they sequester iron during pathogenic infections and release iron for erythropoiesis. Importantly, immunometabolic resetting in macrophages is increasingly becoming linked to their handling of systemic iron recycling by regulating the unidirectional flux of nontransferrin-bound iron (NBTI/non-Tf-Fe 3+ ) and erythrophagocytosis driven by CD163, CD91, and CD47 receptors. Therefore, iron overload can be highly toxic and needs to be strictly governed to maintain iron homeostasis [164,165]. Macrophages have also been highly implicated in metabolic disorders such as T2DM and neurodegenerative diseases, as iron-dependent lipid peroxidation can cause oxidative damage in these diseases via Fenton chemistry. The crosstalk between iron and immune cells (wherein iron trafficking is regulated by cytokines and acute phase proteins) and the iron-dependent cell fate determination of macrophages and lymphocytes during inflammation underscore the requirement for the tight control of iron metabolism and homeostasis [166][167][168][169][170][171][172][173].

Iron Metabolism and Homeostasis-Can Ferroptosis Be the Game Changer?
The authors of a recent case-control study from our health care center in India on COVID-19 patients with and without mucormycosis compared the baseline iron parameters including iron, ferritin, total iron-binding capacity, and percentage transferrin saturation. However, these iron indices did not reveal any significant differences between CAM survivors and non-survivors [174], indicating that much needs to be understood regarding the complexity of iron biology during infectious pathologies such as CAM. Iron or ironcontaining complexes, such as heme and iron-sulfur (Fe-S) clusters are integral to several biological processes such as oxygen transport (hemoglobin), oxygen storage (myoglobin), bioenergetic pathway intermediates (cytochrome-c), metabolic pathway intermediates (amino acid oxidases, fatty acid desaturases, and lipoxygenases), cellular detoxification (cytochrome P450), and cellular defense (nitric oxide synthase, NADPH oxidase, and myeloperoxidase). Notably, the cellular homeostatic machinery is highly reliant upon the cellular redox status, which is determined by the total cellular output of the ROS and antioxidant system. Iron availability is central in maintaining the cellular redox state because it is available in two highly interconvertible forms: reduced Fe 2+ (soluble) and oxidized Fe 3+ (insoluble). Iron cofactors such as heme, iron-sulfur clusters (Fe-S), and iron-oxo systems participate in a wide range of biological processes in electron transfer reactions and are central to metabolic pathways, mitochondrial function, cell cycle, and more [175][176][177][178][179][180][181]. The use of ferritin-bound iron is a cells' protective mechanism of storing iron in macrophages and hepatic cells. Ferritin-bound iron release is a tightly regulated process known as ferritinophagy that involves the nuclear receptor coactivator 4 (NCOA4)mediated transfer of ferritin to autolysosomes, as well as degradation and iron release for the biosynthetic pathways [182].
The overt production of ROS via chemically and reactive Fe 2+ ions is detrimental to cellular membranes because it oxidizes membrane phospholipids and results in membrane rupture and release of DAMPs, which trigger sterile inflammation in iron-dependent regulated cell death known as ferroptosis. Ferroptosis comprises a rapidly evolving research field with enormous therapeutic potential, as it is becoming increasingly linked to inflammatory diseases, infectious diseases, and inflammation-associated immunosuppression. Ferroptosis can be induced by extrinsic (the inhibition of membrane cystine/glutamate antiporter system XC − ) or intrinsic pathways (the inhibition of anti-oxidant glutathione peroxidase/GPX4). Though the terminal effectors of ferroptosis are not clearly understood, it is a form of iron-dependent, peroxide-mediated regulated cell death (RCD) that is distinct from other RCD modes including apoptosis, necroptosis, and pyroptosis because it is independent of the activities of the caspases (apoptosis), MLKL (necroptosis), and gasdermin D (pyroptosis) molecular effectors [183][184][185][186][187][188][189][190][191][192][193][194][195][196].
Ferroptosis has been increasingly linked to the regulation of inflammatory networks in innate immune cells such as neutrophils and macrophages that exhibit graded polarities and differential sensitivities to ferroptosis [197], with pro-inflammatory M1 macrophages marked by the secretion of high iNOS, cytokines, and lipid mediators displaying ferroptoticresistant phenotypes, as opposed to pro-ferroptotic propensity of pro-resolving alternatively activated M2 macrophages that secrete anti-inflammatory molecules. The intracellular redox regulation driven by metabolic iron, thiols, and lipid peroxides is emerging as a new mode of cellular reprograming under the term "redox-stress" that may have potential implications in the resetting of pro-inflammatory pathways driven by the ferroptotic mode of cell death, which is marked by the loss/rupture of the oxidation sensitive phospholipid plasma membranes followed by the release of intracellular DAMPs, resulting in the propagation of sterile inflammation. This is in striking contrast to the anti-inflammatory apoptotic mode of cell death that is marked by the preservation of cell membrane integrity and the containment of intracellular contents that are cleared by hydrolytic digestion and phagocytic engulfment by macrophages [198,199]. Iron-regulatory processes tend to have cell-specific mechanisms [200,201]. Iron-overload-triggered ferroptosis can be inhibited by iron chelators such as deferoxamine, deferiprone, and ciclopirox that decrease intracellular iron levels [202]. Ferroptosis has become increasingly linked to bacterial and yeast infections [203][204][205], but further detailed investigations are warranted in many contexts before targeting ferroptosis as a feasible strategy in infectious pathologies. Nevertheless, emerging opportunities for anti-ferroptotic agents in neurological disease settings are already in progress.

Pro-Ferroptotic Labile Iron Pool (LIP) and RNA-Binding Proteins (RBPs) in Regulation of Ferroptosis and Diabetes
The reactive iron-free radicals in the LIP can oxidatively damage the plasma membrane unsaturated lipids (PUFAs) [175,206]; therefore, the LIP is intrinsically under tight control by the action of iron-buffering compounds such as iron-glutathione (Fe-GSH) complexes (cytosolic GSH occurring at 2-10 mM and complexing Ferrous ions occurring at 1:1 stoichiometry) [207] and iron chaperones. Fe-GSH complexes are reported to be chaperoned by the poly(rC)-binding protein (PCBP) family that dictates intracellular iron distribution and transport [208]. PCBPs (1/2) chaperone iron (at low micromolar affinity) to and from iron transporters (DMT1, ferroportin 1), iron storage pools of ferritin, and iron-containing prolyl hydroxylase (PHD), deoxyhypusine hydroxylase (DOHH), heme-oxygenase (HO-1), and BolA2. PCBPs are RNA-binding proteins (RBPs) with intrinsically disordered regions (IDRs) composed of hnRNP-K-homology (KH) domains that preferably bind to Fe-GSH and facilitate iron transport to client proteins. As the affinities of Fe-GSH complexes and PCBP1 coordination lie in sub-micromolar ranges, it has been suggested that the majority of the cytosolic LIP exists in the PCBP1-Fe-GSH format [199,207]. Therefore, RBPs such as PCBPs intracellularly regulate the chemical reactivity and trafficking of pro-ferroptotic LIP. Ferritin sequesters iron in large amounts as inert ferric oxyhydroxides that can be mobilized by the cells in a process of ferritin degradation via lysosomes or ferritinophagy [209,210] (which is marked by the binding of ferritin to its cognate autophagic cargo receptor NCOA4), and the consequently liberated iron is transported back to the cytosol or mitochondria (erythroblasts) to begin heme synthesis; ferritinophagy and ferroptosis are accordingly implicated in the management of metabolic diseases [211]. PCBP1-and NCOA4-driven ferritinophagy could mobilize large pools of stored iron that may have pro-ferroptotic roles via the delivery of PCBP1-mediated iron to client ferroptotic proteins. PCBP1 chaperones can deliver ferrous ions to mono-or di-iron centers such as HIF-1α (pro-inflammatory macrophages are marked by mitochondrial ROS production and HIF-1α stabilization [212]), the regulating PHD2 enzyme (mono iron center), and the DOHH monooxygenase enzyme (di-iron center), which participates in the hydroxylation of the modified amino acid hypusine that is exclusively present in the eIF5a translational initiation factor. Deoxyhypusine synthase (DHPS)-driven eIF5A hypusination was recently shown to be a feature of proinflammatory macrophages, and DHPS deletion in macrophages resulted in improved insulin sensitivity and glucose homeostasis. Furthermore, hypusination is being increasingly implicated in metabolic diseases and microbial infections [213,214], hypusine polyamine precursors have been reported in intestinal epithelial renewal and M2 macrophage polarization [215], and dietary spermidine was recently shown to boost eIF5A hypusination and protect mitochondrial dysfunction during brain aging [216]. The polyamine pathway intermediate spermidine was recently reported to induce anti-inflammatory pathways following release from apoptotic cells [217]; it has protective effects in metabolic diseases and has emerged as a well-tolerable caloric restriction mimetic and provider of nitric oxide and arginine bioavailability [218]. Interestingly, the SAT1 (spermidine N1 acetyltransferase) gene was recently reported as a metabolic transcription target for p53 and a pro-ferroptotic molecule resulting in lipid peroxidation upon ROS stress [219]. Targeting the polyamine metabolic pathway was reported as a promising approach to control viral infections including SAR-CoV-2 [220]. SAT1 is a catabolic enzyme in the polyamine pathway that leads to the conversion of anti-inflammatory spermidine to N1 acetylspermidine. We recently observed the upregulation and downregulation of N1 acetylspermidine in COVID-19 and CAM plasma samples, respectively, via unbiased metabolomic profiling (unpublished data). Additionally, SAT1 and other ferroptotic pathway genes were differentially regulated in our in vitro culture model that recapitulated the disease modeling of COVID-19 pathways (Figure 6), thereby warranting detailed investigation to understand the role of metabolic ferroptotic and polyamine pathway intermediates in infectious diseases such as COVID-19 and CAM. Amino acid metabolism modifications, such as hypusination, require further investigations to understand their role in regulating infectious and metabolic diseases.
infectious diseases such as COVID-19 and CAM. Amino acid metabolism modifications, such as hypusination, require further investigations to understand their role in regulating infectious and metabolic diseases. Figure 6. Proteolytic stress coupled to growth factor responsiveness is marked by downregulation of ferroptotic pathway genes (highlighted in red), indicating that resistance to ferroptosis could have a beneficial effect in controlling diabetes-associated COVID-19 and CAM. Iron overload is associated with peroxidation of unsaturated fatty acids in plasma membrane, resulting in ferroptotic cell death, inflammation, production of DAMPs, and propagation of inflammatory pathways. Therefore, downregulation of pro-ferroptotic genes could be a strategy to handle iron-overloadmediated inflammatory deregulation in diabetes-associated COVID-19 severity and prevent the spread of CAM. The KEGG pathway was published with slight modifications with permission from Kanehisa laboratory.
The authors of merging studies are trying to leverage post-transcriptional control of immune responses that is regulated by RBPs [130] such as tristetraprolin, Regnase-1, Roquin, and RNA methylases, which coordinate the inflammatory networks in immune cells and associated niches by modulating the mRNA pool of immune pathway genes [221]. Interestingly, PCBP1 RBP was recently implicated as an intracellular checkpoint for shaping T-cell responses by promoting T effector immune response as opposed to T regulatory functions [222]. Diabetic endotheliopathy (possibly driven by the HBP deregulation of endothelial glycocalyx) also posttranscriptionally alters endothelial gene expression via RBPs that are becoming increasingly linked to posttranscriptional immune regulation; RBPs generated during mRNA processing are emerging as frontiers in the regulation of pancreatic beta cell function [223][224][225].
We propose a posttranscriptional gene silencing (RNAi) regulation of TCR genes in COVID-19 pathology, which may intersect with tRNA biology pathway enzymes such as tRNA aminoacyl synthetases that are emerging as important players of immunometabolic dysregulation and linked to the intergenerational inheritance of metabolic traits in diabetes mellitus [226][227][228]. Figure 6. Proteolytic stress coupled to growth factor responsiveness is marked by downregulation of ferroptotic pathway genes (highlighted in red), indicating that resistance to ferroptosis could have a beneficial effect in controlling diabetes-associated COVID-19 and CAM. Iron overload is associated with peroxidation of unsaturated fatty acids in plasma membrane, resulting in ferroptotic cell death, inflammation, production of DAMPs, and propagation of inflammatory pathways. Therefore, downregulation of pro-ferroptotic genes could be a strategy to handle iron-overload-mediated inflammatory deregulation in diabetes-associated COVID-19 severity and prevent the spread of CAM. The KEGG pathway was published with slight modifications with permission from Kanehisa laboratory.
The authors of merging studies are trying to leverage post-transcriptional control of immune responses that is regulated by RBPs [130] such as tristetraprolin, Regnase-1, Roquin, and RNA methylases, which coordinate the inflammatory networks in immune cells and associated niches by modulating the mRNA pool of immune pathway genes [221]. Interestingly, PCBP1 RBP was recently implicated as an intracellular checkpoint for shaping T-cell responses by promoting T effector immune response as opposed to T regulatory functions [222]. Diabetic endotheliopathy (possibly driven by the HBP deregulation of endothelial glycocalyx) also posttranscriptionally alters endothelial gene expression via RBPs that are becoming increasingly linked to posttranscriptional immune regulation; RBPs generated during mRNA processing are emerging as frontiers in the regulation of pancreatic beta cell function [223][224][225].
We propose a posttranscriptional gene silencing (RNAi) regulation of TCR genes in COVID-19 pathology, which may intersect with tRNA biology pathway enzymes such as tRNA aminoacyl synthetases that are emerging as important players of immuno-metabolic dysregulation and linked to the intergenerational inheritance of metabolic traits in diabetes mellitus [226][227][228].

Routes of Infection and Current Perspectives in Clinical Presentation and Diagnosis
There is temporal variability, i.e., seasonal, etiological, geographical, and ecological, in the epidemiology of the transmission of Mucorales of human importance. Frequent hospital outbreaks of cutaneous mucormycosis involving contaminated surgical dressing, adhesive bandages, and tongue depressors have been reported. Furthermore, high spore counts in hospital environments (especially in the ongoing expansion and construction of premises) and contaminated air-conditioners together account for nearly 9% of nosocomial mucormycosis [229]. Regarding CAM, much speculation has been focused on ventilators, catheters, air humidifier bottles, industrial oxygen cylinders, steam inhalation practices, and the extensive usage of masks, which warrant systematic aero-mycological and surface mycological investigations. Furthermore, the Centers for Disease Control (CDC), Atlanta, GA, USA, published a systematic tool that aids in performing environmental assessments when investigating healthcare-associated disease outbreaks due to fungi including Mucorales [6,20,230].
The first step in the diagnosis of CAM is the knowledge of warning symptoms and signs, as well as a high index of clinical suspicion, that includes the identification of DM with or without DKA, the use of SC therapy; the use of immunosuppressants such as tocilizumab; identification of immunodeficiency; iatrogenic suppression using mechanical ventilation; and the identification of hemato-oncological patients, bone-marrow/solid organ transplantation recipients, iron overload, and obviously COVID-19 in the present context. The extent of damage in ROCM or PM needs a multidisciplinary investigation and management with swift decisions from a team of experts, e.g., those in the otorhinolaryngology, ophthalmology, pulmonology, radiology and imaging, internal medicine, infectious disease, neurology, neurosurgery, anesthesiology, maxillofacial surgery, mycology/microbiology, pathology, and clinical pharmacy fields [28,231,232].
In radiological studies using magnetic resonance imaging (MRI) of the paranasal sinuses, contrast-enhanced MRI has been used to identify the infection of vital tissue and assess cerebral invasion in ROCM, diagnostic nasal endoscopy, and color Doppler ultrasonography studies. The computed tomography (CT) of the thorax and the plain and high-resolution (HR) CT for PM have also been used [231,232]. Clinical and radiological findings have overlapped with COVID-19, so the diagnosis of disseminated and pulmonary mucormycosis is challenging. The reverse halo in peripheral lung locations that is suggestive of pulmonary mucormycosis in IC patient(s) and serves as a useful indicator for pre-emptive antifungal therapy is not an indicator for COVID-19, as it can be a commonly overlapping finding. Recently, EORTC and MSG updated the criteria for proven invasive fungal disease (IFD) [231], with recommendations for senso stricto among patients with cancer, transplant recipients, and other severely IC hosts. Even though the diagnosis of IFD via radiological findings remains the gold standard, helping in early detection and swift course of actions, it lacks sensitivity. The most significant pulmonary radiological features of IFD in non-cancer patients and among the trio of invasive pulmonary aspergillosis (IPA), mucormycosis, and COVID-19 overlap with "atypical" non-nodular pattern, consolidation, and ground-glass opacities [231].
The laboratory diagnosis and confirmation of mucormycosis is easy if the biopsy samples are available, as in the case of ROCM, whereas the diagnosis of disseminated or pulmonary mucormycosis is difficult due to the nonavailability of biopsy specimens due to various limitations. The microscopic examination of the tissues using KOH mount and Calcofluor white aids the rapid confirmation of diagnosis. Under microscopy, the Mucorales hyphae classically appears hyaline, broad (2-8 µm diameter), ribbon-like (often folded), pauci-septate, or aseptate with a right-angled branching. Unlike other molds, Mucorales grow faster within 24-48 h and appear cottony. However, the sensitivity of cultures is not more than 80%. The species-level identification of cultures can be made with conventional phenotypic, morphological, and physiological features such as growth rate and incubation temperatures on various media, but it needs expertise in mycological techniques and identification. The method of choice for the species identification of Mucorales is therefore ITS sequencing, which is mostly restricted to reference laboratories. Even though ITS barcoding has been a preferred molecular marker for the identification of various Mucorales species, it has a few limitations such as the failure to differentiate R. microsporus and R. azygosporus because both belong to the same species [233].
Therefore, attempts to directly diagnose mucormycosis from clinical specimens have been made by standardizing the Mucorales PCR using conventional, nested, RFLP, and real-time PCR (qPCR) formats on blood samples and other matrices such as bronchoalveolar lavage (BAL) or tissue specimens, and several in-house assays have shown good sensitivity. The commercially available multiplex, pan-Mucorales real-time (qRCR) kit-MicroGenius assay by PathoNostics readily detects several Mucorales species including Rhizopus, Lichtheimia, Rhizomucor, and Cunninghamella in sera and BAL, thus showing promising rapid diagnostic utility in present clinical setups. In this era of rapid diagnosis, lower turnaround-time (TAT), and laboratory quality initiatives, several technologies such as matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) have increasingly gained momentum and will start influencing the clinical microbiology laboratories in the future. The negative results of the serological markers such as (1→3)-β-D-glucan (BDG) and galactomannan (GM) (used in other IFD) can aid the diagnosis of invasive mucormycosis (IM) during strong suspicion of IFD. The evaluation of these surrogate markers in proven IM using an enzyme-linked immunospot (ELISpot) assay (leveraging Mucorales-specific T-cells with a derived diagnostic cut-off) has been reported, and elevated IL-10 and IL-4 levels in symptomatic patients and elevated Mucorales-specific T-cells with a higher IFN-γ levels in non-symptomatic patients were observed. These results indicate the assay's promising utility in hematological patients and warrant rigorous investigation into its potential as a surrogate marker in IM and other patient populations [234].

Clinical Perspective of Rhino-Orbital Mucormycosis and Patient Management
Mucormycosis, primarily caused by Mucorales in immunocompromised or immunecompetent hosts with diabetes/trauma, is a critical scenario for every clinician dealing with this condition. Rhino-orbital mucormycosis is the most common form of the disease; its fulminant nature, the extensive angio-invasion-induced ischemic necrosis-related inflammation of tissues, delays in suspicion in primary care, and lack of effective medical antifungal therapy have worsened diagnosis challenges.
Rhino-orbito-cerebral mucormycosis patients present with a history of facial heaviness, fever, purulent nasal discharge, headache, nasal congestion, and sinus pain. The disease can progress and present with the destruction of the turbinates and the nasal septum, paleness, blackening in the nasal and palatal mucosa, and ulcer formation in the palate with exposed underlying osteomyelitic bone. Peri-orbital swelling decreases vision, proptosis, and ophthalmoplegia with or without diplopia in patients of orbital involvement, and it alters neurological function and consciousness in cases of cerebral involvement, thus indicating ominous disease aggressiveness. Diagnosis primarily rests on microbiological analysis with KOH wet mounts of scrapping or tissue samples taken from pale, necrotic, and inflamed areas. Radiological imaging with CT/MRI and histological analysis of tissue samples aid diagnosis.
A diagnosis or high clinical suspicion of diagnosis leads to management that primarily involves the following steps:

•
Continued treatment of the primary immune deficiency condition. • Correction of biochemical parameters and management of associated diabetic status.

•
Aggressive debridement of the necrotic tissues of the rhino-orbital-facial region with the aim to clear all necrotic tissue and osteomyelitic bones to the maximum extent until the tissues bleed, with caution used in case of cerebral involvement to not to debride brain tissue. • Post-debridement wound and cavity local care and adjuvant medical management with amphotericin B and/or posaconazole and isavucanazole.
• Continuation of cavity care after the completion of therapy with the regular clearance of crusts and saline irrigations for 3-6 months after treatment. • Prosthetic rehabilitation and/or reconstruction of the defect.
The morbidities and functional compromises associated with disease and management lead to significant losses of productivity and hinder the quality of life of patients, especially during the current COVID-19 pandemic, in which instances of mucormycosis have increased to overwhelming levels due to underlying immune dysregulation by COVID-19 and associated steroid therapies. Hence, high clinical suspicion and early referrals are key to early management and the preservation of the quality of life in cases of rhino-orbital mucormycosis.

Future Perspective-A Working Hypothesis to Explain Development of COVID-19 and CAM in the Backdrop of Diabetes
Serine proteases can have cell-autonomous functions (besides SARS-CoV-2 activation) in olfactory microenvironment, in which they can activate and signal through cognate GPCR family of protease-activated receptors (PARs) that participate in transducing downstream inflammatory and calcium signaling pathways, resulting in the regulation of ion channels and transporters. In diabetic patients, endotheliopathy in the vascular olfactory niche can further add to the PAR-transducing protease repertoire by offering pre-activated TF-FVIIa and FXa serine proteases that could have Janus faces during inflammatory propagation. TF-FVIIa and FXa can activate PAR signaling concomitant to the complement coagulation pathway intensification that is central player in COVID-19 pathogenesis [235], and the resultant inflammatory loops can have pro-fibrogenic functions as the associated plasmin and uPAR-uPA signaling modulators are known to have growth factor-binding, TGF-beta-activating, integrin-signaling, and ECM-remodeling features. We report that protease stress is associated with concomitant metabolic deregulation and the differential expression of olfactory transduction pathway genes including the olfactory/odorant receptors (ORs). Importantly, the majority of the olfactory receptors are orphan receptors, and deorphanization is as a major challenge. Many fatty acid and lipid metabolites are potential activating and inhibitory ligands of ORs, and emerging studies have suggesting a broader expression pattern of ORs beyond the olfactory epithelium, including adipocytes, airways, kidneys, livers, lungs, and adipocytes. Extra-nasal ORs have been reported to perform seminal functions including blood pressure regulation via the renin-angiotensinaldosterone pathway in kidneys and the hypoxia-sensing ORs in the glomus cells of the carotid body. Hypoxia metabolite lactate is a partial agonist of carotid body ORs. Hepatic ORs regulate hepatic lipid accumulation via the regulation of GLP-1, resulting in glucose and insulin tolerance. Airway smooth muscle cells express ORs that regulate bronchoconstriction through calcium signaling via the cAMP pathway. However, ligand-dependent contractility effects that are linked to inflammatory pathways of IL-8 and GMCSF have also been reported. The adipocytic effects of ORs are mediated by hepatic fatty acid oxidation and brown adipose tissue thermogenesis. Epicardial adipose tissue and diabetes mellitus patient plasma contain medium chain fatty acids that can activate cardiac muscle cells and regulate ionotropic effects [236,237]. The appreciable overlap of ORs expression pattern with affected organs in COVID-19 and the OR regulation of RAAS and hypoxia functions led us to propose their potential contribution in disease pathology and their use as therapeutic targets for diabetes, COVID-19 pathology, and CAM.
Importantly, neural-crest-originating astrocytes and pericytes are becoming increasingly linked to underlying neurovascular abnormalities in COVID-19 pathobiology. We also observed the upregulation of neural crest pathways in protease-treated monocytic cells. It is a possibility that developmental epithelial-mesenchymal transition (EMT) events initiated by active proteases during embryonic neural-crest induction could be recapitulated by providing exogenous proteolytic exposure to pathology competent monocytic cells in vitro, resulting in the upregulation of developmental multipotent neural-crest-like stem cells with ectomesodermal potential that capable of upregulating neurogenesis/gliogenesis (neurons and astrocyte) and vasculogenesis (microvascular pericyte), crucial cellular processes underlying neuroinflammatory diseases [Sharma et al., unpublished data]. The OR-expressing sustentacular cells in olfactory epithelia are glia-like, and the neuroendocrine cells have also been reported to be neural-crest-derived; their participation in the lung neuroinflammation in context of ARDS has been proposed with the pharmacological neuromodulation of the vagus nerve as a potential therapeutic approach to treat COVID-19 [238]. The hypothalamus-pituitary-adrenal gland axis (HPA), along with the vagal reflex and the possible involvement of the carotid body, has been proposed for the neural control of inflammation wherein the deregulated vagal anti-inflammatory reflex is central to inflammation and metabolic diseases including type 2 diabetes. The HPA axis under stress releases cortisol-releasing hormones, which induce the secretion of anti-inflammatory glucocorticoids from adrenal glands. Vagal anti-inflammatory reflex sense inflammation and the activation of efferent vagus nerve fibers results in dampened cytokine production following the binding of neurotransmitter acetylcholine to its receptor nicotinic α7nAChR on immune cells such as macrophages. Furthermore, the efferent sympathetic output to adrenal gland medulla chromaffin (neuroendocrine) cells triggers epinephrine release to activate specific receptors in immune cells to resolve inflammation [239]. Carotid body chemoreceptors have been proposed as afferent arms of the anti-inflammatory reflex and are becoming increasingly linked to inflammatory diseases such as sepsis and intermittent hypoxia [240]. The glomus cells of the carotid body that act as hypoxia sensors bear olfactory receptors and are neural-crest-derived, so we hypothesize carotid body chemosensors as central players in driving neuroinflammatory events in COVID-19/post-COVID neurological sequelae including anosmia.
Notably, small molecule neurotransmitters and neuromodulators generally belong to the class of amino acid metabolites including glutamate, sulfur-containing cysteine and methionine, proline, GABA, lysine, arginine, glycine, serine, alanine, aromatic amino acids, and branched-chain amino acids. These amino acid neurotransmitters recycle between neurons and astrocytes and conduct nerve impulse transmission through ionotropic channels, and a few of these amino acids participate in generating mitochondrial oxidative and nitrosative stress. The aromatic amino acid tyrosine is the precursor for the dopamine, norepinephrine, and epinephrine neurotransmitters that regulate neuroinflammation and are implicated in many neurocognitive diseases [241]. We hypothesize that the oxidative and nitrosative stress in macrophages due to metabolic deregulation during diabetes and COVID-19 can alter iron homeostasis and therefore lead to the lipid peroxidation and ferroptosis that compromise macrophage functionality and anti-Mucorales prowess. Vascular macrophages have been shown to upregulate ORs that bind to lipid metabolites and activate inflammasome and IL-1 production [242]. The parallel deregulation of the olfactory transduction pathway via metabolic-stress-triggered lipid and amino acid metabolites (proposed as potential ligands for ORs) and the activation of PARs due to serine proteases can drive the co-deregulation of olfactory transduction and complement coagulation pathways in macrophages (vascular immune) and olfactory epithelium (neurosensory) cells causing anosmia and neuroinflammation. The subsequent generation of DAMPs, as reported during inflammasome activation, can be passed onto the astrocytes and pericytes of the brain, which results in neurocognitive and neurodegenerative features. The post-viral effects of COVID-19 in the olfactory system and their neurological connections were described recently [69]. The pre-symptomatic or acute phase of COVID-19 is marked by high viral loads/replication in the nasal epithelium, which makes it a potential target to inhibit the intensification of infection by limiting the spread of the infection. Therefore, the prophylactic/therapeutic targeting of the olfactory epithelium by employing intranasal sprays could be a potential approach to prevent diabetes-associated COVID-19 and CAM (Figure 7). by limiting the spread of the infection. Therefore, the prophylactic/therapeutic targeting of the olfactory epithelium by employing intranasal sprays could be a potential approach to prevent diabetes-associated COVID-19 and CAM (Figure 7).

Conclusions
The mechanistic insights of chemosensory/neurosensory deregulation in the olfactory epithelium and the attendant immune landscape reshaping during COVID-19 infection remain largely unexplored due to infection control concerns in disease-active patients. We leveraged the proteolytic predominance of olfactory microenvironment during acute phase of the disease to develop a novel virus-free cellular model to identify a potential host-associated serine protease-interactome that displays COVID-19-like disease signatures including maturity-onset diabetes, complement-coagulation, and olfactory transduction. Our data support the role of olfactory dysfunction in predicting neurological sequelae in CNS and suggest a new paradigm that could involve olfactory transduction-mediated neuroinflammation in the respiratory tract and peripheral tissues with resident macrophages expressing neuropeptide receptors. We also gathered appreciable patient data from COVID-19 and CAM plasma metabolomics and proteomics that suggest the beneficial effects of rescuing olfactory epithelium health with the potential repurposing of anti-metabolic drugs such as 2DG (2-deoxy-D-glucose) and nutraceutical azoles with anti-inflammatory and anti-oxidative effects. Our preliminary data demonstrated the anti-fungal activity of 2DG and L-carnosine (growth inhibition assay) against clinically relevant Mucorales species, a novel anti-fungal action (unpublished data). Both drugs have proven anti-diabetic and potential in-silico anti-COVID-19 effects, so our next work is directed towards the development of novel We acknowledge SARS-CoV-2, ACE2, and TMPRSS2 receptor image credits as adapted from an image by Davian Ho for the Innovative Genomics Institute." https://innovativegenomics.org/free-covid-19-illustrations/ (accessed on 2 January 2022) [244].

Conclusions
The mechanistic insights of chemosensory/neurosensory deregulation in the olfactory epithelium and the attendant immune landscape reshaping during COVID-19 infection remain largely unexplored due to infection control concerns in disease-active patients. We leveraged the proteolytic predominance of olfactory microenvironment during acute phase of the disease to develop a novel virus-free cellular model to identify a potential host-associated serine protease-interactome that displays COVID-19-like disease signatures including maturity-onset diabetes, complement-coagulation, and olfactory transduction. Our data support the role of olfactory dysfunction in predicting neurological sequelae in CNS and suggest a new paradigm that could involve olfactory transduction-mediated neuroinflammation in the respiratory tract and peripheral tissues with resident macrophages expressing neuropeptide receptors. We also gathered appreciable patient data from COVID-19 and CAM plasma metabolomics and proteomics that suggest the beneficial effects of rescuing olfactory epithelium health with the potential repurposing of anti-metabolic drugs such as 2DG (2-deoxy-D-glucose) and nutraceutical azoles with anti-inflammatory and anti-oxidative effects. Our preliminary data demonstrated the anti-fungal activity of 2DG and L-carnosine (growth inhibition assay) against clinically relevant Mucorales species, a novel anti-fungal action (unpublished data). Both drugs have proven anti-diabetic and potential in-silico anti-COVID-19 effects, so our next work is directed towards the development of novel intranasal anti-diabetic sprays for controlling infectious airway diseases such as COVID-19 and CAM (Sharma et al., unpublished work).